Therapeutic compounds and compositions

ABSTRACT

The present invention provides compounds and compositions that inhibit Factor XIa or kallikrein and methods of using these compounds and composition.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/290,565, filedOct. 11, 2016, which is a continuation of U.S. Ser. No. 14/614,169,filed Feb. 4, 2015, which claims priority under 35 U.S.C. § 119(e) toU.S. provisional patent application U.S. Ser. No. 61/937,031, filed Feb.7, 2014, the contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Blood coagulation is the first line of defense against blood lossfollowing injury. The blood coagulation “cascade” involves a number ofcirculating serine protease zymogens, regulatory cofactors andinhibitors. Each enzyme, once generated from its zymogen, specificallycleaves the next zymogen in the cascade to produce an active protease.This process is repeated until finally thrombin cleaves thefibrinopeptides from fibrinogen to produce fibrin that polymerizes toform a blood clot. Although efficient clotting limits the loss of bloodat a site of trauma, it also poses the risk of systemic coagulationresulting in massive thrombosis. Under normal circumstances, hemostasismaintains a balance between clot formation (coagulation) and clotdissolution (fibrinolysis). However, in certain disease states such asacute myocardial infarction and unstable angina, the rupture of anestablished atherosclerotic plaque results in abnormal thrombusformation in the coronary arterial vasculature.

Diseases that stem from blood coagulation, such as myocardialinfarction, unstable angina, atrial fibrillation, stroke, pulmonaryembolism, and deep vein thrombosis, are among the leading causes ofdeath in developed countries. Current anticoagulant therapies, such asinjectable unfractionated and low molecular weight (LMW) heparin andorally administered warfarin (coumadin), carry the risk of bleedingepisodes and display patient-to-patient variability that results in theneed for close monitoring and titration of therapeutic doses.Consequently, there is a large medical need for novel anticoagulationdrugs that lack some or all of the side effects of currently availabledrugs.

Factor XIa is an attractive therapeutic target involved in the pathwayassociated with these diseases. Increased levels of Factor XIa or FactorXIa activity have been observed in several thromboembolic disorders,including venous thrombosis (Meijers et al., N. Engl. J. Med. 342:696,2000), acute myocardial infarction (Minnema et al., Arterioscler ThrombVasc Biol 20:2489, 2000), acute coronary syndrome (Butenas et al.,Thromb Haemost 99:142, 2008), coronary artery disease (Butenas et al.,Thromb Haemost 99:142, 2008), chronic obstructive pulmonary disease(Jankowski et al., Thromb Res 127:242, 2011), aortic stenosis (BloodCoagul Fibrinolysis, 22:473, 2011), acute cerebrovascular ischemia(Undas et al., Eur J Clin Invest, 42:123, 2012), and systolic heartfailure due to ischemic cardiomyopathy (Zabcyk et al., Pol Arch MedWewn. 120:334, 2010). Patients that lack Factor XI because of a geneticFactor XI deficiency exhibit few, if any, ischemic strokes (Salomon etal., Blood, 111:4113, 2008). At the same time, loss of Factor XIaactivity, which leaves one of the pathways that initiate coagulationintact, does not disrupt hemostasis. In humans, Factor XI deficiency canresult in a mild-to-moderate bleeding disorder, especially in tissueswith high levels of local fibrinolytic activity, such as the urinarytract, nose, oral cavity, and tonsils. Moreover, hemostasis is nearlynormal in Factor XI-deficient mice (Gailani, Blood Coagul Fibrinolysis,8:134, 1997). Consequently, compounds that inhibit Factor XIa have thepotential to prevent or treat a wide range of thromboembolic disorderswhile avoiding the side effects and therapeutic challenges that plaguedrugs that inhibit other components of the coagulation pathway.Moreover, due to the limited efficacy and adverse side effects of somecurrent therapeutics for the inhibition of undesirable thrombosis (e.g.,deep vein thrombosis and stroke), improved compounds and methods (e.g.,those associated with Factor XIa) are needed for preventing or treatingundesirable thrombosis.

Another therapeutic target is the enzyme kallikrein. Human plasmakallikrein is a serine protease that may be responsible for activatingseveral downstream factors (e.g., bradykinin and plasmin) that arecritical for coagulation and control of e.g., blood pressure,inflammation, and pain. Kallikreins are expressed e.g., in the prostate,epidermis, and the central nervous system (CNS) and may participate ine.g., the regulation of semen liquefaction, cleavage of cellularadhesion proteins, and neuronal plasticity in the CNS. Moreover,kallikreins may be involved in tumorigenesis and the development ofcancer and angioedema, e.g., hereditary angioedema. Overactivation ofthe kallikrein-kinin pathway can result in a number of disorders,including angioedema, e.g., hereditary angioedema (Schneider et al., J.Allergy Clin. Immunol. 120:2, 416, 2007). To date, there are limitedtreatment options for HAE (e.g., WO2003/076458). As such, therapeuticsare needed for preventing or treating these diseases.

SUMMARY OF THE INVENTION

The present invention features compounds that inhibit Factor XIa orkallikrein and methods for preventing or treating undesired thrombosisor angiodema (e.g., hereditary angiodema) by administering one or moreof these compounds alone or in combination with other molecules to amammal. The invention also provides methods for designing or selectingadditional Factor XIa or kallikrein inhibitors using these structures.Desirably, these compounds have certain structural, physical, andspatial characteristics that enable the compounds to interact withspecific residues of the active site of Factor XIa or kallikrein.

In one aspect, the present invention is directed to a compound offormula (I):

or a pharmaceutically acceptable salt thereof, wherein R¹ is H or —C₁₋₆alkyl; R² is H, —C₁-₆ alkyl, —CO₂R⁵, —C(O)NR⁹R¹⁰, —CN, —SO_(q)R⁵, —OR⁵,—CHN(OR⁵), or a heteroaryl; R³ is H or —C₁-C₆ alkyl; A is a bond, C₁₋₆alkylene, C₂₋₆ alkenylene, or C₂₋₆ alkynylene; R⁴ is cycloalkyl, aryl,heteroaryl or heterocyclyl (e.g., piperidonyl, piperidinyl, pyridonyl,benzodioxolyl e.g., difluorobenzodioxolyl), each of which is substitutedwith 0-3 occurrences of R⁶; each R⁵ is independently H, —C₁₋₆ alkyl,aralkyl, or aryl substituted with 0-3 occurrences of —NH₂ or R⁶; each R⁶is independently halo, hydroxy, cyano, nitro, —C₁₋₆ alkyl (e.g., methyl,ethyl, haloalkyl (e.g., —CF ₃)), —C₁₋₆ alkoxy (e.g., haloalkoxy (e.g.,—OCF ₃)), —NHR¹⁰, —NR⁹R¹⁰, —C(O)R¹¹, —C(O)OR¹¹, —C(O)NR⁹R¹⁰,—C(NR⁸)(N(R⁸)₂), —SO_(q)R¹¹, —SO₂NR⁹R¹⁰, —NHC(O)OR¹¹, NHC(O)R¹¹,—OC(O)R¹¹, aryl, heteroaryl, aralkyl, cycloalkyl, heteroaralkyl,heterocyclyl, or heterocyclylalkyl (e.g.,

or two R⁶ groups together with the atoms to which they are attached forma 5-7-membered ring (e.g.,

X is —C(O)O—, —OC(O)—, —C(O)S(O)₂—, —S(O)₂C(O)—, —C(O)N(R⁵)— or—N(R⁵)C(O)—; Y is —C₁₋₆ alkyl, cycloalkyl (e.g., 3 to 8-memberedcycloalkyl, e.g., 5 to 7-membered cycloalkyl), aryl, heteroaryl, orheterocyclyl (e.g., 3 to 8-membered heterocyclyl, e.g., 5 to 7-memberedheterocyclyl), each of which is substituted with 0-3 occurrences of —NH₂or R⁶; R⁷ is H, —C₁₋₆ alkyl (e.g., haloalkyl (e.g., —CF₃)), cycloalkyl,aryl, heteroaryl, or heterocyclyl, each of which is substituted with 0-3occurrences of —NH₂ or R⁶; each R⁸ is independently H, —C₁₋₆ alkyl(e.g., haloalkyl (e.g., —CF ₃)), —C(O)R⁵, —C(O)OR⁵, aryl, heteroaryl,aralkyl, heteroaralkyl, heterocyclyl, or heterocyclylalkyl; each of R⁹and R¹° is independently —C₁₋₆ alkyl, cycloalkyl, heterocyclyl, aryl, orheteroaryl, or R⁹ and R¹⁰ together form an optionally substituted5-7-membered ring (e.g.,

each R¹¹ is independently H, —C₁₋₆ alkyl (e.g., substituted alkyl (e.g.,

aralkyl, or aryl; q is an integer from 0 to 2; and n is an integer from0 to 2.

In some embodiments, R¹ is H.

In some embodiments, R² is —CO₂R⁵ and R⁵ is H, —C₁₋₆ alkyl, aralkyl, oraryl substituted with 1 occurrence of —NH₂ or R⁶. In some embodiments,R⁵ is H, methyl, ethyl, isopropyl, or benzyl substituted with 1occurrence of R⁶. In some embodiments, R⁵ is H. In some embodiments, R⁵is ethyl.

In some embodiments, A is C₁₋₆ alkylene (e.g., ethylene or propylene).

In some embodiments, R⁴ is aryl or heteroaryl. In some embodiments, R⁴is phenyl with 0 occurrences of R⁶. In some embodiments, R⁴ is phenylsubstituted with 1-2 occurrences of R⁶. In some embodiments, R⁶ is halo,—C₁₋₆ alkoxy or —C(NR⁸)(N(R⁸)₂). In some embodiments, R⁶ is—C(NR⁸)(N(R⁸)₂) and each R⁸ is H. In some embodiments, R⁶ is—C(NR⁸)(N(R⁸)₂) and each R⁸ is independently H or —C(O)OR⁵. In someembodiments, R⁶ is —C(NR⁸)(N(R⁸)₂) and R⁵ is —C₁₋₆ alkyl (e.g., hexyl).In some embodiments, R⁴ is heteroaryl (e.g., a 6-membered heteroaryl or5-membered heteroaryl) substituted with 0-3 occurrences of R⁶. In someembodiments, R⁴ is a 6-membered heteroaryl (e.g., pyridyl) substitutedwith 0-3 occurrences of R⁶. In some embodiments, R⁴ is anitrogen-containing heteroaryl (e.g., pyridyl). In some embodiments, R⁴is pyridyl substituted with 1-2 occurrences of R⁶. In some embodiments,R⁶ is halo (e.g., chloro, bromo, fluoro). In some embodiments, R⁶ is—NHR¹⁰ and R¹⁰ is —C₁₋₆ alkyl. In some embodiments, R⁶ is —NHC(O)OR¹¹,and R¹¹ is —C₁₋₆ alkyl (e.g., substituted alkyl (e.g.,

In some embodiments, X is —C(O)N(R⁵)— or —N(R⁵)C(O)—. In someembodiments, X is —C(O)N(R⁵)— and R⁵ is H.

In some embodiments, n is 0.

In some embodiments, R⁷ is —C₁₋₆ alkyl (e.g., methyl, ethyl, propyl,—CF₃).

In some embodiments, Y is cycloalkyl (e.g., cyclohexyl). In someembodiments, Y is aryl or heteroaryl substituted with 0-3 occurrences ofR⁶. In some embodiments, Y is phenyl substituted with 0 occurrences ofR⁶. In some embodiments, Y is phenyl substituted with 1 occurrence ofR⁶. In some embodiments, Y is phenyl substituted with 2 occurrences ofR⁶.

In some embodiments, Y is aryl or heteroaryl and R⁶ is haloalkoxy (e.g.,—OCF₃). In some embodiments, Y is aryl or heteroaryl and two R⁶ groupstaken together with the atoms to which they are attached form a 5-7membered ring. In some embodiments, Y is aryl or heteroaryl and two R⁶groups taken together with the atoms to which they are attached form a5-7 membered ring selected from:

In some embodiments, Y is phenyl and R⁶ is haloalkoxy (e.g., —OCF₃). Insome embodiments, Y is phenyl and two R⁶ groups taken together with theatoms to which they are attached form a 5-7 membered ring. In someembodiments, Y is phenyl and two R⁶ groups taken together with the atomsto which they are attached form a 5-7 membered ring selected from:

In some embodiments, the compound of formula (I) is selected from acompound of formula (Ia):

wherein R¹, R², R³, R⁴, R⁷, and Y are as described for formula (I), andm is an integer from 1 to 6.

In some embodiments, the compound of formula (Ia) is selected from acompound of formula (Ib):

wherein R¹, R², R³, R⁴, R⁷, Y and m are as described for formula (Ia).

In some embodiments, the compound of formula (Ib) is:

In one aspect, the present invention is directed to a compound offormula (II):

or a pharmaceutically acceptable salt thereof, wherein R¹ is H or —C₁₋₆alkyl; R² is H, —C₁₋₆ alkyl, —CO₂R⁵, —C(O)NR⁹R¹⁰, —CN, —SO_(q)R⁵, —OR⁵,—CHN(OR⁵) or a heteroaryl; R³ is H or —C₁₋₆ alkyl; A is a bond, C₁₋₆alkylene, C₂₋₆ alkenylene or C₂₋₆ alkynylene; R⁴ is cycloalkyl, aryl,heteroaryl or heterocyclyl, each of which is substituted with 0-3occurrences of —NH₂ or R⁶; each R⁵ is independently H, —C₁₋₆ alkyl,aralkyl, or aryl substituted with 0-3 occurrences of —NH₂ or R⁶; each R⁶is independently halo, hydroxy, cyano, nitro, —C₁₋₆ alkyl (e.g., methyl,ethyl, haloalkyl (e.g., —CF₃)), C₁₋₆ alkoxy (e.g., haloalkoxy (e.g.,—OCF₃)), —NR⁹R¹⁰, —NHR¹⁰, —C(O)R¹¹, —C(O)OR¹¹, —C(O)NR⁹R¹⁰,—C(NR⁸)(N(R⁸)₂), —SO_(q)NR¹¹, —SO₂NR⁹R¹⁰, —NHC(O)OR¹¹, —NHC(O)R¹¹, aryl,heteroaryl, aralkyl, cycloalkyl, heteroaralkyl, heterocyclyl orheterocyclylalkyl, or two R⁶ groups together with the atoms to whichthey are attached form a 5-7-membered ring (e.g.,

X is —C(O)O—, —OC(O)—, —C(O)S(O)₂—, —S(O)₂C(O)—, —C(O)N(R⁵)— or—N(R⁵)C(O)—; Y is cycloalkyl (e.g., 3 to 8-membered cycloalkyl, e.g., 5to 7-membered cycloalkyl), heteroaryl, or heterocyclyl (e.g., 3 to8-membered heterocyclyl, e.g., 5 to 7-membered heterocyclyl), each ofwhich is substituted with 0-3 occurrences of —NH₂ or R⁶; or substituted—C₁₋₆ alkyl or substituted aryl (e.g., substituted with 1-3 R⁶); R⁷ isH, —C₁₋₆ alkyl (e.g., methyl, haloalkyl (e.g., —CF₃)), cycloalkyl, aryl,heteroaryl or heterocyclyl, each of which is substituted with 0-3occurrences of —NH₂ or R⁶; each R⁸ is independently H, —C₁₋₆ alkyl(e.g., haloalkyl (e.g., —CF ₃)), —C(O)R⁵, —C(O)OR⁵, aryl, heteroaryl,aralkyl, heteroaralkyl, heterocyclyl or heterocyclylalkyl; each of R⁹and R¹⁰ is independently —C₁₋₆ alkyl, cycloalkyl, heterocyclyl, aryl, orheteroaryl, or R⁹ and R¹⁰ together form an optionally substituted5-7-membered ring; each R¹¹ is independently H, —C₁₋₆ alkyl (e.g.,substituted alkyl (e.g.,

aralkyl, or aryl; q is an integer from 0 to 2; and n is an integer from0 to 2.

In some embodiments, R¹ is H.

In some embodiments, R² is —CO₂R⁵ and R⁵ is H, —C₁₋₆ alkyl, aralkyl, oraryl substituted with 1 occurrence of —NH₂ or R⁶. In some embodiments,R⁵ is H, methyl, ethyl, isopropyl, or benzyl substituted with 1occurrence of R⁶. In some embodiments, R⁵ is H. In some embodiments, R⁵is ethyl.

In some embodiments, A is C₁₋₆ alkylene (e.g., ethylene or propylene).

In some embodiments, R⁴ is aryl or heteroaryl. In some embodiments, R⁴is phenyl substituted with 1 occurrence of R⁶. In some embodiments, R⁶is halo, C₁₋₆ alkoxy or —C(NR⁸)(N(R⁸)₂). In some embodiments, R⁶ is—C(NR⁸)(N(R⁸)₂) and each R⁸ is H. In some embodiments, R⁸ isindependently H or —CO₂R⁵. In some embodiments, R⁵ is —C₁₋₆ alkyl (e.g.,hexyl). In some embodiments, R⁴ is heteroaryl (e.g., a 6-memberedheteroaryl or 5-membered heteroaryl) substituted with 0-3 occurrences of—NH₂ or R⁶. In some embodiments, R⁴ is a 6-membered heteroaryl (e.g.,pyridyl) substituted with 0-3 occurrences of —NH₂ or R⁶. In someembodiments, R⁴ is a nitrogen-containing heteroaryl (e.g., pyridyl)substituted with 0-3 occurrences of —NH₂ or R⁶. In some embodiments, R⁴is pyridyl substituted with 1 occurrence of R⁶. In some embodiments, R⁶is halo (e.g., chloro, bromo, fluoro). In some embodiments, R⁴ ispyridyl substituted with 1 occurrence of —NH₂. In some embodiments, R⁶is —NHR¹⁰ and R¹⁰ is —C₁₋₆ alkyl. In some embodiments, R⁶ is—NHC(O)OR¹¹, and R¹¹ is —C₁₋₆ alkyl (e.g., substituted alkyl (e.g.,

In some embodiments, X is —C(O)N(R⁵)— or —N(R⁵)C(O)—. In someembodiments, X is —C(O)N(R⁵)— and R⁵ is H.

In some embodiments, n is 0.

In some embodiments, R⁷ is —C₁₋₆ alkyl (e.g., methyl, ethyl, propyl,—CF₃). In some embodiments, R⁷ is methyl. In some embodiments, R⁷ is—CF₃.

In some embodiments, Y is cycloalkyl, heteroaryl, or heterocyclyl, eachof which is substituted with 0-3 occurrences of —NH₂ or R⁶, orsubstituted aryl. In some embodiments, Y is cycloalkyl (e.g.,cyclohexyl). In some embodiments, Y is heteroaryl (e.g., pyridyl,pyrazinyl, pyrimidinyl, quinolinyl, isoquinolinyl, thiazolyl,indazolyl). In some embodiments, Y is substituted aryl (e.g.,substituted phenyl, naphthyl). In some embodiments, Y is substitutedphenyl substituted with 1-2 occurrences of R⁶. In some embodiments, Y isphenyl substituted with 1 occurrence of R⁶. In some embodiments, Y isphenyl and R⁶ is haloalkoxy (e.g., —OCF₃). In some embodiments, Y isphenyl and R⁶ is halo (e.g., chloro, bromo, fluoro). In someembodiments, Y is phenyl substituted with 2 occurrences of R⁶.

In some embodiments, R⁶ is haloalkoxy (e.g., —OCF₃). In someembodiments, two R⁶ groups taken together with the atoms to which theyare attached form a 5-7 membered ring. In some embodiments, two R⁶groups taken together with the atoms to which they are attached form a5-7 membered ring and the ring is selected from:

In some embodiments, the compound of formula (II) is selected from acompound of formula (IIa):

wherein R¹, R², R³, R⁴, R⁷, and Y are as described for formula (II), andm is an integer from 1 to 6.

In some embodiments, the compound of formula (IIa) is selected from acompound of formula (IIb):

wherein R¹, R², R³, R⁴, R⁷, Y and m are as described for formula (IIa).

In some embodiments, the compound of formula (IIb) is:

In some embodiments, the compound of formula (IIb) is selected from acompound of formula (IIc):

In one aspect, the present invention is directed to a compound offormula (III):

or a pharmaceutically acceptable salt thereof, wherein R¹ is H or —C₂₋₆alkyl; R² is H, —C₂₋₆ alkyl, haloalkyl, —CO₂R¹², —C(O)NH₂, —CN,—SO_(q)R⁵, —OR⁵, —CHN(OR⁵), or a heteroaryl; R³ is H or —C₁₋₆ alkyl; Ais a bond, C₁₋₆ alkylene, C₂₋₆ alkenylene or C₂₋₆ alkynylene; R⁴ iscycloalkyl, aryl, heteroaryl or heterocyclyl, each of which issubstituted with 0-3 occurrences of —NH₂ or R⁶; each R⁵ is independentlyH, —C₁₋₆ alkyl, aralkyl, or aryl substituted with 0-3 occurrences of—NH₂ or R⁶; each R⁶ is independently halo, hydroxy, cyano, nitro, —C₁₋₆alkyl, —C₁₋₆ alkoxy, —NHR¹⁰, —NR⁹R¹⁰, —C(O)OR¹¹, —C(O)NR⁹R¹⁰,—C(NR⁸)(N(R⁸)₂), —SO_(q)R¹¹, —SO₂NR⁹R¹⁰, —NHC(O)OR¹¹, —NHC(O)R¹¹, aryl,heteroaryl, aralkyl, cycloalkyl, heteroaralkyl, heterocyclyl orheterocyclylalkyl, or two R⁶ groups together with the atoms to whichthey are attached form a 5-7-membered ring; X is —C(O)O—, —OC(O)—,—C(O)S(O)₂—, —S(O)₂C(O)—, —C(O)N(R⁵)— or —N(R⁵)C(O)—; Y is —C₁₋₆ alkyl,cycloalkyl, aryl, heteroaryl, or heterocyclyl, each of which issubstituted with 0-3 occurrences of —NH₂ or R⁶; R⁷ is H, —C₁₋₆ alkyl,cycloalkyl, aryl, heteroaryl or heterocyclyl, each of which issubstituted with 0-3 occurrences of —NH₂ or R⁶; each R⁸ is independentlyH, —C₁₋₆ alkyl, —C(O)R⁵, —C(O)OR⁵, aryl, heteroaryl, aralkyl,heteroaralkyl, heterocyclyl, or heterocyclylalkyl; each of R⁹ and R¹⁰ isindependently —C₁₋₆ alkyl, cycloalkyl, heterocyclyl, aryl, orheteroaryl, or R⁹ and R¹⁰ together form an optionally substituted5-7-membered ring; each R¹¹ is independently H, —C₁₋₆ alkyl, aralkyl, oraryl; each R¹² is independently haloalkyl, optionally substituted —C₃₋₆alkyl, or aralkyl; q is an integer from 0 to 2; and n is an integer from0 to 2.

In some embodiments, R¹ is H.

In some embodiments, R² is haloalkyl (e.g., —CH₂F, —CHF₂, —CF₃),—CO₂R¹², —C(O)NH₂, —CN, —SO_(q)R⁵ (e.g., —SO₂R⁵), —OR⁵, —CHN(OR⁵), or aheteroaryl (e.g., triazolyl, tetrazolyl, optionally substitutedoxazolyl, optionally substituted isoxazolyl). In some embodiments, R¹²is haloalkyl, propyl, or aralkyl (e.g., benzyl). In some embodiments, R⁵is H, —C₁₋₆ alkyl (e.g., methyl, ethyl, propyl) or aryl (e.g., phenyl).

In some embodiments, A is C₁₋₆ alkylene (e.g., ethylene or propylene).

In some embodiments, R⁴ is aryl or heteroaryl. In some embodiments, R⁴is phenyl substituted with 1 occurrence of R⁶. In some embodiments, R⁶is C₁₋₆ alkoxy or —C(NR⁸)(N(R⁸)₂). In some embodiments, R⁶ is—C(NR⁸)(N(R⁸)₂) and each R⁸ is H. In some embodiments, R⁸ isindependently H or —C(O)OR⁵. In some embodiments, R⁵ is —C₁₋₆ alkyl(e.g., hexyl).

In some embodiments, R⁴ is heteroaryl (e.g., a 6-membered heteroaryl or5-membered heteroaryl) substituted with 0-3 occurrences of —NH₂ or R⁶.In some embodiments, R⁴ is a 6-membered heteroaryl (e.g., pyridyl)substituted with 0-3 occurrences of —NH₂ or R⁶. In some embodiments, R⁶is halo (e.g., chloro). In some embodiments, R⁴ is pyridyl substitutedwith 1 occurrence of —NH₂.

In some embodiments, X is —C(O)N(R⁵)— or —N(R⁵)C(O)—. In someembodiments, X is —C(O)N(R⁵)— and R⁵ is H.

In some embodiments, n is 0.

In some embodiments, R⁷ is —C₁₋₆ alkyl (e.g., methyl). In someembodiments, R⁷ is aryl (e.g., phenyl).

In some embodiments, Y is cycloalkyl, aryl, heteroaryl, or heterocyclyl,each of which is substituted with 0-3 occurrences of —NH₂ or R⁶. In someembodiments, Y is phenyl substituted with 0 occurrences of R⁶. In someembodiments, Y is phenyl substituted with 1 occurrence of R⁶. In someembodiments, of R⁶ is —C₁₋₆ alkoxy.

In some embodiments, the compound of formula (III) is selected from acompound of formula (IIIa):

wherein R¹, R², R³, R⁴, R⁷, and Y are as described for formula (III),and m is an integer from 1 to 6.

In some embodiments, the compound of formula (IIIa) is selected from acompound of formula (IIIb):

wherein R¹, R², R³, R⁴, R⁷, Y and m are as described for formula (IIIa).

In some embodiments, the compound of formula (IIIb) is:

In one aspect, the present invention is directed to a compound offormula (IV):

or a pharmaceutically acceptable salt thereof, wherein R¹ is H or —C₁₋₆alkyl; R² is H, —C₁₋₆ alkyl, —CO₂R⁵, —C(O)NR⁹R¹⁰, —CN, —SO_(q)R⁵, —OR⁵,—CHN(OR⁵) or a heteroaryl; R³ is H or —C₁₋₆ alkyl; A is a bond, C₁₋₆alkylene, C₂₋₆ alkenylene or C₂₋₆ alkynylene; R⁴ is cycloalkyl, aryl,heteroaryl or heterocyclyl, each of which is substituted with 0-3occurrences of —NH₂ or R⁶; each R⁵ is independently H, —C₁₋₆ alkyl,aralkyl, or aryl substituted with 0-3 occurrences of —NH₂ or R⁶; each R⁶is independently halo, —C₁₋₆ alkyl, —C₁₋₆ alkoxy, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)OR¹¹, —C(O)NR⁹R¹⁰, —C(NR⁸)(N(R⁸)₂), —SO_(q)R¹¹, —SO₂NR⁹R¹⁰,—NHC(O)OR¹¹, aryl, heteroaryl, aralkyl, cycloalkyl, heteroaralkyl,heterocyclyl or heterocyclylalkyl, or two R⁶ groups together with theatoms to which they are attached form a 5-7-membered ring; X is —C(O)O—,—OC(O)—, —C(O)S(O)₂—, —S(O)₂C(O)—, —C(O)N(R⁵)— or —N(R⁵)C(O)—; Y is H,—C₁₋₆ alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl, each ofwhich is substituted with 0-3 occurrences of —NH₂ or R⁶; R⁷ is H, —C₂₋₆alkyl, haloalkyl, cycloalkyl, heteroaryl or heterocyclyl, each of whichis substituted with 0-3 occurrences of —NH₂ or R⁶, or substituted aryl;each R⁸ is independently H, —C₁₋₆ alkyl, —C(O)R⁵, —C(O)OR⁵, aryl,heteroaryl, aralkyl, heteroaralkyl, heterocyclyl or heterocyclylalkyl;each of R⁹ and R¹⁰ is independently —C₁₋₆ alkyl, cycloalkyl,heterocyclyl, aryl, or heteroaryl, or R⁹ and R¹⁰ together form anoptionally substituted 5-7-membered ring; each R¹¹ is independently H,—C₁₋₆ alkyl, aralkyl, or aryl; q is an integer from 0 to 2; and n is aninteger from 0 to 2.

In some embodiments, R¹ is H.

In some embodiments, R² is —CO₂R⁵, wherein R⁵ is H or —C₁₋₆ alkyl (e.g.,ethyl).

In some embodiments, A is C₁₋₆ alkylene (e.g., ethylene or propylene).

In some embodiments, R⁴ is aryl or heteroaryl. In some embodiments, R⁴is phenyl substituted with 1 occurrence of R⁶. In some embodiments, R⁶is halo, C₁₋₆ alkoxy or —C(NR⁸)(N(R⁸)₂). In some embodiments, R⁶ is—C(NR⁸)(N(R⁸)₂) and each R⁸ is H. In some embodiments, R⁴ is heteroaryl(e.g., a 6-membered heteroaryl or 5-membered heteroaryl) substitutedwith 0-3 occurrences of —NH₂ or R⁶. In some embodiments, R⁴ is a6-membered heteroaryl (e.g., pyridyl) substituted with 0-3 occurrencesof —NH₂ or R⁶. In some embodiments, R⁶ is halo (e.g., chloro, bromo,fluoro). In some embodiments, R⁶ is —NHC(O)OR¹¹, and R¹¹ is —C₁₋₆ alkyl(e.g., substituted alkyl (e.g.,

In some embodiments, X is —C(O)N(R⁵)— or —N(R⁵)C(O)—. In someembodiments, X is —C(O)N(R⁵)— and R⁵ is H.

In some embodiments, R⁷ is H, —C₂₋₆ alkyl, haloalkyl, cycloalkyl, orsubstituted aryl.

In some embodiments, n is 0.

In some embodiments, Y is H, —C₁₋₆ alkyl, cycloalkyl, aryl, heteroaryl,or heterocyclyl, each of which is substituted with 0-3 occurrences of—NH₂ or R⁶. In some embodiments, Y is aryl (e.g., aryl, naphthyl). Insome embodiments, Y is phenyl substituted with 0 occurrences of R⁶. Insome embodiments, Y is phenyl substituted with 1 occurrence of R⁶. Insome embodiments, R⁶ is haloalkoxy (e.g., —OCF₃) or halo (e.g., chloro).In some embodiments, Y is aryl or heteroaryl and two R⁶ groups takentogether with the atoms to which they are attached form a 5-7 memberedring selected from:

In some embodiments, Y is phenyl and two R⁶ groups taken together withthe atoms to which they are attached form a 5-7 membered ring. In someembodiments, Y is phenyl and two R⁶ groups taken together with the atomsto which they are attached form a 5-7 membered ring selected from:

In some embodiments, Y is heteroaryl (e.g., pyridyl, pyrazinyl,pyrimidinyl, quinolinyl, isoquinolinyl, thiazolyl, indazolyl)substituted with 0-3 occurrences of R⁶. In some embodiments, Y iscycloalkyl (e.g., cyclohexyl). In some embodiments, Y is —C₁₋₆ alkylsubstituted with 0 occurrences of R⁶. In some embodiments, Y is —C₁₋₆alkyl substituted with 1-3 occurrences of R⁶, and R⁶ is aryl. In someembodiments, Y is —C₁₋₆ alkyl substituted with 2 occurrences of R⁶, andR⁶ is aryl (e.g., phenyl).

In some embodiments, the compound of formula (IV) is selected from acompound of formula (IVa):

wherein R¹, R², R³, R⁴, R⁷, and Y are as described for formula (IV), andm is an integer from 1 to 6.

In some embodiments, the compound of formula (IVa) is selected from acompound of formula (IVb):

wherein R¹, R², R³, R⁴, R⁷, Y and m are as described for formula (IVa).

In some embodiments, the compound of formula (IV) is:

In one aspect, the present invention is directed to a compound offormula (V):

or a pharmaceutically acceptable salt thereof, wherein R¹ is H or —C₁₋₆alkyl; R² is H, —C₁₋₆ alkyl, —CO₂R⁵, —C(O)NR⁹R¹⁰, —CN, —SO_(q)R⁵, —OR⁵,—CHN(OR⁵) or a heteroaryl; R³ is H or —C₁₋₆ alkyl; A is —C₂₋₆ alkylene,—C₂₋₆ alkenylene, or —C₂₋₆ alkynylene; R⁴ is cycloalkyl, aryl,heteroaryl or heterocyclyl, each of which is substituted with 0-3occurrences of —NH₂ or R⁶; each R⁵ is independently H, —C₁₋₆ alkyl,aralkyl, or aryl substituted with 0-3 occurrences of —NH₂ or R⁶; each R⁶is independently halo, hydroxy, cyano, nitro, —C₁₋₆ alkyl, —C₁₋₆ alkoxy,—NHR¹⁰, —NR⁹R¹⁰, —C(O)R¹¹, —C(O)OR¹¹, —C(O)NR⁹R¹⁰, —C(NR⁸)(N(R⁸)₂),—SO_(q)R¹¹, —SO₂NR⁹R¹⁰, —NHC(O)OR¹¹, —NHC(O)R¹¹, aryl, heteroaryl,aralkyl, cycloalkyl, heteroaralkyl, heterocyclyl or heterocyclylalkyl,or two R⁶ groups together with the atoms to which they are attached forma 5-7-membered ring; X is —C(O)O—, —OC(O)—, —C(O)S(O)₂—, —S(O)₂C(O)—,—C(O)N(R⁵)— or —N(R⁵)C(O); Y is —C₁₋₆ alkyl, cycloalkyl, aryl,heteroaryl, or heterocyclyl, each of which is substituted with 0-3occurrences of —NH₂ or R⁶; R⁷ is H, —C₁₋₆ alkyl, cycloalkyl, aryl,heteroaryl or heterocyclyl, each of which is substituted with 0-3occurrences of —NH₂ or R⁶; each R⁸ is independently H, —C₁₋₆ alkyl,—C(O)R⁵, —C(O)OR⁵, aryl, heteroaryl, aralkyl, heteroaralkyl,heterocyclyl or heterocyclylalkyl; each of R⁹ and R¹⁰ is independently—C₁₋₆ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, or R⁹ andR¹⁰ together form an optionally substituted 5-7-membered ring; each R¹¹is independently H, —C₁₋₆ alkyl, aralkyl, or aryl; q is an integer from0 to 2; and n is an integer from 0 to 2.

In some embodiments, R¹ is H.

In some embodiments, R² is —CO₂R⁵, wherein R⁵ is H, —C₁₋₆ alkyl (e.g.,ethyl), or aralkyl (e.g., benzyl).

In some embodiments, A is C₂₋₆ alkylene (e.g., ethylene or propylene).

In some embodiments, R⁴ is aryl or heteroaryl. In some embodiments, R⁴is aryl. In some embodiments, R⁴ is phenyl substituted with 1 occurrenceof R⁶. In some embodiments, R⁶ is —C₁₋₆ alkyl, halo (e.g., chloro,bromo, fluoro), haloalkoxy (e.g., —OCF₃) or —C(NR⁸)(N(R⁸)₂). In someembodiments, R⁶ is —C(NR⁸)(N(R⁸)₂) and each R⁸ is H. In someembodiments, R⁶ is —C(NR⁸)(N(R⁸)₂) and each R⁸ is H. In someembodiments, R⁸ is H or —C(O)OR⁵. In some embodiments, R⁵ is —C₁₋₆ alkyl(e.g., hexyl).

In some embodiments, R⁴ is heteroaryl (e.g., a 6-membered heteroaryl or5-membered heteroaryl) substituted with 0-3 occurrences of —NH₂ or R⁶.In some embodiments, R⁴ is a 6-membered heteroaryl (e.g., pyridyl)substituted with 0-3 occurrences of —NH₂ or R⁶. In some embodiments, R⁶is halo (e.g., chloro).

In some embodiments, X is —C(O)N(R⁵)— or —N(R⁵)C(O)—. In someembodiments, X is —C(O)N(R⁵)— and R⁵ is H.

In some embodiments, n is 0.

In some embodiments, R⁷ is —C₁₋₆ alkyl (e.g., methyl, —CF₃).

In some embodiments, Y is —C₁₋₆ alkyl (e.g., ethyl, propyl), cycloalkyl,aryl, heteroaryl, or heterocyclyl, each of which is substituted with 0-3occurrences of —NH₂ or R⁶. In some embodiments, Y is cycloalkyl (e.g.,cyclohexyl). In some embodiments, Y is phenyl substituted with 0occurrences of R⁶. In some embodiments, Y is phenyl substituted with 1occurrence of R⁶. In some embodiments, R⁶ is —C₁₋₆ alkyl, halo (e.g.,chloro, bromo, fluoro) or haloalkoxy (e.g., —OCF₃). In some embodiments,Y is phenyl substituted with 2 occurrences of R⁶. In some embodiments,two R⁶ groups taken together with the atoms to which they are attachedform a 5-7 membered ring. In some embodiments, two R⁶ groups takentogether with the atoms to which they are attached form a 5-7 memberedring and the ring is selected from:

In some embodiments, the compound of formula (V) is selected from acompound of formula (Va):

wherein R¹, R², R³, R⁴, R⁷, and Y are as described for formula (V), andm is an integer from 2 to 6.

In some embodiments, the compound of formula (Va) is selected from acompound of formula (Vb):

wherein R¹, R², R³, R⁴, R⁷, Y and m are as described for formula (Va).

In some embodiments, the compound of formula (Vb) is:

In one aspect, the present invention is directed to a compound offormula (VI):

or a pharmaceutically acceptable salt thereof, wherein R¹ is H or —C₁₋₆alkyl; R² is H, —C₁₋₆ alkyl, —CO₂R⁵, —C(O)NR⁹R¹⁰, —CN, —SO_(q)R⁵, —OR⁵,—CHN(OR⁵) or a heteroaryl; R³ is H or —C₁₋₆ alkyl; R⁴ is cycloalkyl,aryl, heteroaryl or heterocyclyl, each of which is substituted with 0-3occurrences of —NH₂ or R⁶; each R⁵ is independently H, —C₁₋₆ alkyl,aralkyl, or aryl substituted with 0-3 occurrences of —NH₂ or R⁶; each R⁶is independently halo, hydroxy, cyano, nitro, —C₁₋₆ alkyl, —C₁₋₆ alkoxy,—NHR¹⁰, —NR⁹R¹⁰, —C(O)R¹¹, —C(O)OR¹¹, —C(O)NR⁹R¹⁰, —C(NR⁸)(N(R⁸)₂),—SO_(q)R¹¹, —SO₂NR⁹R¹⁰, —NHC(O)OR¹¹, —NHC(O)R¹¹, aryl, heteroaryl,aralkyl, cycloalkyl, heteroaralkyl, heterocyclyl or heterocyclylalkyl,or two R⁶ groups together with the atoms to which they are attached forma 5-7-membered ring; Y is —C₁-₆ alkyl, cycloalkyl, aryl, heteroaryl, orheterocyclyl, each of which is substituted with 0-3 occurrences of —NH₂or R⁶; R⁷ is H, —C₁₋₆ alkyl, cycloalkyl, aryl, heteroaryl orheterocyclyl, each of which is substituted with 0-3 occurrences of —NH₂or R⁶; each R⁸ is independently H, —C₁₋₆ alkyl, —C(O)R⁵, —C(O)OR⁵, aryl,heteroaryl, aralkyl, heteroaralkyl, heterocyclyl or heterocyclylalkyl;each of R⁹ and R¹⁰ is independently —C₁₋₆ alkyl, cycloalkyl,heterocyclyl, aryl, or heteroaryl, or R⁹ and R¹⁰ together form anoptionally substituted 5-7-membered ring; each R¹¹ is independently H,—C₁₋₆ alkyl, aralkyl, or aryl; R¹³ is —C₁₋₆ alkyl; q is an integer from0 to 2; and m is an integer from 1 to 6.

In some embodiments, R¹ is H.

In some embodiments, R² is —CO₂R⁵, wherein R⁵ is H or —C₁₋₆ alkyl (e.g.,ethyl).

In some embodiments, A is —C₁₋₆ alkylene (e.g., ethylene or propylene).

In some embodiments, R⁴ is aryl or heteroaryl. In some embodiments, R⁴is phenyl substituted with 1 occurrence of R⁶. In some embodiments, R⁶is halo (e.g., chloro).

In some embodiments, R⁴ is heteroaryl (e.g., a 6-membered heteroaryl or5-membered heteroaryl) substituted with 0-3 occurrences of —NH₂ or R⁶.In some embodiments, R⁴ is a 6-membered heteroaryl (e.g., pyridyl)substituted with 0-3 occurrences of —NH₂ or R⁶. In some embodiments, R⁶is halo (e.g., chloro). In some embodiments, R⁴ is a pyridyl substitutedwith 1 occurrence of —NH₂.

In some embodiments, X is —C(O)N(R⁵)— or —N(R⁵)C(O)—. In someembodiments, X is —C(O)N(R⁵)— and R⁵ is H.

In some embodiments, n is 0.

In some embodiments, R⁷ is —C₁₋₆ alkyl (e.g., methyl).

In some embodiments, Y is cycloalkyl, aryl, heteroaryl, or heterocyclyl,each of which is substituted with 0-3 occurrences of —NH₂ or R⁶. In someembodiments, Y is cycloalkyl (e.g., cyclohexyl). In some embodiments, Yis phenyl substituted with 0 occurrences of R⁶. In some embodiments, Yis phenyl substituted with 1 occurrence of R⁶.

In some embodiments, R¹³ is methyl.

In some embodiments, the compound of formula (VI) is selected from acompound of formula (VIa):

wherein R¹, R², R³, R⁴, R⁷, and Y are as described for formula (VI), andm is an integer from 1 to 6.

In some embodiments, the compound of formula (VIa) is selected from acompound of formula (VIb):

wherein R¹, R², R³, R⁴, R⁷, Y and m are as described for formula (VIa).

In some embodiments, the compound of formula (VIb) is:

In one aspect, the present invention is directed to a compound offormula (VII):

or a pharmaceutically acceptable salt thereof, wherein R¹ is H or —C₁₋₆alkyl; R² is H, —C₁₋₆ alkyl, —CO₂R⁵, —C(O)NR⁹R¹⁰, —CN, —SO_(q)R⁵, —OR⁵,—CHN(OR⁵) or a heteroaryl; R³ is —C₁₋₆ alkyl; A is a bond, C₁₋₆alkylene, C₂₋₆ alkenylene or C₂₋₆ alkynylene; R⁴ is cycloalkyl, aryl,heteroaryl or heterocyclyl, each of which is substituted with 0-3occurrences of —NH₂ or R⁶; each R⁵ is independently H, —C₁₋₆ alkyl,aralkyl, or aryl substituted with 0-3 occurrences of —NH₂ or R⁶; each R⁶is independently halo, hydroxy, cyano, nitro, —C₁₋₆ alkyl, —C₁₋₆ alkoxy,—NHR¹⁰, —NR⁹R¹⁰, —C(O)R¹¹, —C(O)OR¹¹, —C(O)NR⁹R¹⁰, —C(NR⁸)(N(R⁸)₂),—SO_(q)R¹¹, —SO₂NR⁹R¹⁰, —NHC(O)OR¹¹, —NHC(O)R¹¹, aryl, heteroaryl,aralkyl, cycloalkyl, heteroaralkyl, heterocyclyl or heterocyclylalkyl,or two R⁶ groups together with the atoms to which they are attached forma 5-7-membered ring; X is —C(O)O—, —OC(O)—, —C(O)S(O)₂—, —S(O)₂C(O)—,—C(O)N(R⁵)— or —N(R⁵)C(O)—; Y is H, —C₁₋₆ alkyl, cycloalkyl, aryl,heteroaryl, or heterocyclyl, each of which is substituted with 0-3occurrences of —NH₂ or R⁶; R⁷ is H, —C₁₋₆ alkyl, cycloalkyl, aryl,heteroaryl or heterocyclyl, each of which is substituted with 0-3occurrences of —NH₂ or R⁶; each R⁸ is independently H, —C₁₋₆ alkyl,—C(O)R⁵, —C(O)OR⁵, aryl, heteroaryl, aralkyl, heteroaralkyl,heterocyclyl or heterocyclylalkyl; each of R⁹ and R¹⁰ is independently—C₁₋₆ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, or R⁹ andR¹⁰ together form an optionally substituted 5-7-membered ring; each R¹¹is independently H, —C₁₋₆ alkyl, aralkyl, or aryl; q is an integer from0 to 2; and n is an integer from 0 to 2.

In some embodiments, R¹ is H.

In some embodiments, R² is —CO₂R⁵, wherein R⁵ is H, —C₁₋₆ alkyl, oraralkyl (e.g., benzyl).

In some embodiments, R³ is methyl.

In some embodiments, A is C₁₋₆ alkylene (e.g., ethylene or propylene).

In some embodiments, R⁴ is aryl or heteroaryl. In some embodiments, R⁴is phenyl substituted with 1 occurrence of R⁶. In some embodiments, R⁶is halo (e.g., chloro) or C₁₋₆ alkoxy.

In some embodiments, R⁴ is heteroaryl (e.g., a 6-membered heteroaryl or5-membered heteroaryl) substituted with 0-3 occurrences of —NH₂ or R⁶.In some embodiments, R⁴ is a 6-membered heteroaryl (e.g., pyridine)substituted with 0-3 occurrences of —NH₂ or R⁶. In some embodiments, R⁶is halo (e.g., chloro).

In some embodiments, X is —C(O)N(R⁵)— or —N(R⁵)C(O)—. In someembodiments, X is —C(O)N(R⁵)— and R⁵ is H.

In some embodiments, n is 0.

In some embodiments, R⁷ is —C₁₋₆ alkyl (e.g., methyl) or aryl (e.g.,phenyl).

In some embodiments, Y is aryl, heteroaryl, or heterocyclyl, each ofwhich is substituted with 0-3 occurrences of —NH₂ or R⁶. In someembodiments, Y is aryl. In some embodiments, Y is phenyl substitutedwith 0 occurrences of R⁶. In some embodiments, Y is phenyl substitutedwith 1 occurrence of R⁶.

In some embodiments, the compound of formula (VII) is selected from acompound of formula (VIIa):

wherein

-   -   R¹, R², R³, R⁴, R⁷, and Y are as described for formula (VII),        and m is an integer from 1 to 6.

In some embodiments, the compound of formula (VIIa) is selected from acompound of formula (VIIb):

wherein R¹, R², R³, R⁴, R⁷, Y and m are as described for formula (VIIa).

In some embodiments, the compound of formula (VIIb) is:

In some embodiments, the compound of formula (VIIb) is selected from acompound of formula (VIIc):

wherein R¹, R², R³, R⁷, Y and m are as described for formula (VIIb), andR⁴ is cycloalkyl, aryl, heteroaryl or heterocyclyl, each of which issubstituted with 0-3 occurrences of R⁶.

In one aspect, the present invention is directed to a pharmaceuticalcomposition comprising a compound of the formula (I)-(VII) or apharmaceutically acceptable salt thereof and one or morepharmaceutically acceptable excipients.

In some embodiments, the composition is provided as a solution.

In one aspect, the present invention is directed to a method of reducingthe risk of stroke (e.g., ischemia, e.g., a transient ischemic event) ina subject that has suffered an ischemic event (e.g., a transientischemic event), comprising administering to the subject an effectiveamount of a compound of the formula (I)-(VII) or a pharmaceuticallyacceptable salt thereof, or of a composition described herein (e.g., acomposition comprising a compound of the formula (I)-(VII)).

In some embodiments, the administering reduces the risk of stroke in asubject as compared to a subject who is not administered with thecompound.

In one aspect, the present invention is directed to a method of reducingnon-central nervous system systemic embolism (e.g., ischemia, e.g., atransient ischemic event) in a subject that has suffered an ischemicevent (e.g., a transient ischemic event), comprising administering tothe subject an effective amount of a compound of the formula (I)-(VII)or a pharmaceutically acceptable salt thereof, or of a compositiondescribed herein (e.g., a composition comprising a compound of theformula (I)-(VII)).

In some embodiments, the administering reduces non-central nervoussystem systemic embolism in a subject as compared to a subject who isnot administered with the compound.

In one aspect, the present invention is directed to a method of treatingdeep vein thrombosis comprising administering to the subject that hassuffered an ischemic event (e.g., a transient ischemic event), aneffective amount of a compound of the formula (I)-(VII) or apharmaceutically acceptable salt thereof, or of a composition describedherein (e.g., a composition comprising a compound of the formula(I)-(VII)).

In one aspect, the present invention is directed to a method of reducingthe risk of recurrence of deep vein thrombosis comprising administeringto the subject that has suffered deep vein thrombosis, an effectiveamount of a compound of the formula (I)-(VII) or a pharmaceuticallyacceptable salt thereof, or of a composition described herein (e.g., acomposition comprising a compound of the formula (I)-(VII)). In someembodiments, the administering reduces the risk of recurrence of deepvein thrombosis in a subject as compared to a subject who is notadministered with the compound.

In one aspect, the present invention is directed to a method of reducingthe risk of recurrence of pulmonary embolism (e.g., symptomaticpulmonary embolism) comprising administering to the subject that hassuffered a pulmonary embolism, an effective amount of a compound of theformula (I)-(VII) or a pharmaceutically acceptable salt thereof, or of acomposition described herein (e.g., a composition comprising a compoundof the formula (I)-(VII)).

In some embodiments, the administering reduces the risk of recurrence ofpulmonary embolism in a subject as compared to a subject who is notadministered with the compound.

In one aspect, the present invention is directed to a method ofprophylaxis of pulmonary embolism in a subject that has suffered apulmonary embolism, comprising administering to the subject an effectiveamount of a compound of the formula (I)-(VII) or a pharmaceuticallyacceptable salt thereof, or of a composition described herein (e.g., acomposition comprising a compound of the formula (I)-(VII)).

In one aspect, the present invention is directed to a method of treatinga subject that has had an ischemic event (e.g., transient ischemia),comprising: administering a compound of the formula (I)-(VII) or apharmaceutically acceptable salt thereof, or of a composition describedherein (e.g., a composition comprising a compound of the formula(I)-(VII)) to the subject. In some embodiments, the compound isadministered to the subject within 24 hours or less, e.g., 12, 10, 9, 8,7, 6 hours or less, after the onset of the ischemic event in thesubject.

In one aspect, the present invention is directed to a method of treatinga subject that has had an ischemic event (e.g., transient ischemia),comprising: administering a compound of the formula (I)-(VII) or apharmaceutically acceptable salt thereof, or of a composition describedherein (e.g., a composition comprising a compound of the formula(I)-(VII)) to the subject. In some embodiments, the compound isadministered to the subject within more than 2 hours to 12 hours, e.g.,more than 2 hours to 10 hours or less, more than 2 hours to 8 hours orless, after the onset of the ischemic event in the subject.

In one aspect, the present invention is directed to a method ofinhibiting Factor XIa in a subject, comprising administering to thesubject that has suffered ischemia an effective amount of a compound ofthe formula (I)-(VII) or a pharmaceutically acceptable salt thereof, orof a composition described herein (e.g., a composition comprising acompound of the formula (I)-(VII)).

In some embodiments, the subject is a mammal (e.g., a human). In someembodiments, the subject is undergoing surgery (e.g., knee replacementsurgery, hip replacement surgery). In some embodiments, the subject is asubject with nonvalvular atrial fibrillation. In some embodiments, thesubject has one or more of the following risk factors for stroke: aprior stroke (e.g., ischemic, unknown, hemorrhagic), transient ischemicattack, or non-CNS systemic embolism. In some embodiments, the subjecthas one or more of the following risk factors for stroke: 75 years orolder of age, hypertension, heart failure or left ventricular ejectionfraction (e.g., less than or equal to 35%), or diabetes mellitus.

In some embodiments, the compound is administered by oral or parenteral(e.g., intravenous) administration.

In some embodiments, the compound is administered prior to an ischemicevent (e.g., to a subject is at risk of an ischemic event).

In some embodiments, the compound is administered after an ischemicevent (e.g., a transient ischemic event).

In some embodiments, the compound is administered about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more after an ischemic event(e.g., a transient ischemic event).

In some embodiments, the compound is administered about 1, 2, 3, 4, 5,6, 7, or 8 weeks or more after an ischemic event (e.g., a transientischemic event).

In some embodiments, the compound is administered in combination with anadditional therapeutic agent. In some embodiments, the additionaltherapeutic agent is administered after administration of the compound.In some embodiments, the additional therapeutic agent is administeredorally. In some embodiments, the additional therapeutic agent isadministered at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, or24 hours or more after administration of the compound. In someembodiments, the additional therapeutic agent is administered at least1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days or more after administration ofthe compound. In some embodiments, the additional therapeutic agent isadministered about 1 day, about 2 days, about 3 days, about 4 days,about 5 days, about 6 days, about 7 days or more after administration ofthe compound.

In some embodiments, the additional therapeutic agent is administeredchronically (e.g., for about 1 day, about 2 days, about 3 days, about 4days, about 5 days, about 6 days, about 7 days, about 8 days, about 9days, about 10 days, about 11 days, about 12 days, about 13 days, orabout 14 days or more) after administration of the compound.

In some embodiments, the additional therapeutic agent treats a sideeffect (e.g., active pathological bleeding or severe hypersensitivityreactions (e.g., anaphylactic reactions), spinal and or epiduralhematoma, gastrointestinal disorder (e.g., abdominal pain upper,dyspepsia, toothache), general disorders and administration siteconditions (e.g., fatigue), infections and infestations (e.g.,sinusitis, urinary tract infection), musculoskeletal and connectivetissues disorders (e.g., back pain, osteoarthritis), respiratory,thoracic and mediastinal disorders (e.g., oropharyngeal pain), injury,poisoning, and procedural complications (e.g., wound secretion),musculoskeletal and connective tissues disorders (e.g., pain inextremity, muscle spasm), nervous system disorders (e.g., syncope), skinand subcutaneous tissue disorders (e.g., pruritus, blister), blood andlymphatic system disorders (e.g., agranulocytosis), gastrointestinaldisorders (e.g., retroperitoneal hemorrhage), hepatobiliary disorders(e.g., jaundice, cholestasis, cytolytic hepatitis), immune systemdisorders (e.g., hypersensitivity, anaphylactic reaction, anaphylacticshock, angioedema), nervous system disorders (e.g., cerebral hemorrhage,subdural hematoma, epidural hematoma, hemiparesis), skin andsubcutaneous tissue disorders (e.g., Stevens-Johnson syndrome).

In some embodiments, the additional therapeutic agent is a NSAID (e.g.,aspirin, naproxen), platelet aggregation inhibitor (e.g., clopidogrel),or anticoagulant (e.g., warfarin, enoxaparin).

In some embodiments, the additional therapeutic agent results in anadditive therapeutic effect.

In some embodiments, the additional therapeutic agent results in asynergistic therapeutic effect.

In another aspect, the invention features a pharmaceutical compositioncomprising a compound described herein (e.g., a compound of formula(I)-(VII)) and a pharmaceutically acceptable excipient.

In another aspect, the invention features a method of modulating (e.g.,inhibiting) Factor XIa in a patient. The method comprises the step ofadministering an effective amount of a compound described herein (e.g.,a compound of formula (I)-(VII)) to a patient in need thereof, therebymodulating (e.g., inhibiting) Factor XIa.

In another aspect, the invention features a method of treating a subjectin need thereof for a thromboembolic disorder. The method comprisesadministering to the subject a therapeutically effective amount of acompound described herein (e.g., a compound of formula (I)-(VII)).

The thromboembolic disorder can be arterial cardiovascularthromboembolic disorders, venous cardiovascular thromboembolicdisorders, and thromboembolic disorders in the chambers of the heart;including unstable angina, an acute coronary syndrome, first myocardialinfarction, recurrent myocardial infarction, ischemia (e.g., ischemicsudden death or transient ischemic attack), stroke, atherosclerosis,peripheral occlusive arterial disease, venous thrombosis, deep veinthrombosis, thrombophlebitis, arterial embolism, coronary arterialthrombosis, cerebral arterial thrombosis, cerebral embolism, kidneyembolism, pulmonary embolism, and thrombosis resulting from (a)prosthetic valves or other implants, (b) indwelling catheters, (c)stents, (d) cardiopulmonary bypass, (e) hemodialysis, or (f) otherprocedures in which blood is exposed to an artificial surface thatpromotes thrombosis.

In another aspect, the invention features a method of treating a subjectidentified as being at risk for stroke or thrombosis thereby reducingthe likelihood of stroke or thrombosis in the subject. In someembodiments, the subject is further identified as being at risk forbleeding (e.g., excessive bleeding) or sepsis. In some embodiments, thetreatment is effective without bleeding liabilities. In someembodiments, the treatment is effective to maintain the patency ofinfusion ports and lines. In addition, the compounds described herein(e.g., compounds of formula (I)-(VII)) are useful in the treatment andprevention of other diseases in which the generation of thrombin hasbeen implicated as playing a physiologic role. For example, thrombin hasbeen implicated in contributing to the morbidity and mortality ofchronic and degenerative diseases, such as cancer, arthritis,atherosclerosis, vascular dementia, and Alzheimer's disease, by itsability to regulate many different cell types through specific cleavageand activation of a cell surface thrombin receptor, mitogenic effects,diverse cellular functions such as cell proliferation, for example,abnormal proliferation of vascular cells resulting in restenosis orangiogenesis, release of PDGF, and DNA synthesis. Inhibition of FactorXIa effectively blocks thrombin generation and therefore neutralizes anyphysiologic effects of thrombin on various cell types. Therepresentative indications discussed above include some, but not all, ofthe potential clinical situations amenable to treatment with a FactorXIa inhibitor.

In another aspect, the invention features a method of treating a subjectthat has edema (e.g., angioedema, e.g., hereditary angioedema),comprising administering a compound of the formula (I)-(VII) or apharmaceutically acceptable salt thereof, or of a composition describedherein (e.g., a composition comprising a compound of the formula(I)-(VII)) to the subject.

In another aspect, the invention features a method of inhibitingkallikrein in a subject, comprising administering to the subject withedema (e.g., angioedema, e.g., hereditary angioedema), an effectiveamount of a compound of the formula (I)-(VII) or a pharmaceuticallyacceptable salt thereof, or of a composition described herein (e.g., acomposition comprising a compound of the formula (I)-(VII)) to thesubject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. IA-1M depict Chart A and the synthesis of Structures A-8 and A-9.

FIG. 2 depicts Chart B and the synthesis of Structure B-5.

FIG. 3 depicts Chart C and D and the synthesis of Structure D-8.

FIG. 4 depicts Chart E and the synthesis of Structure E-6.

FIG. 5 depicts Chart F and the synthesis of Structure F-8.

FIG. 6 depicts Chart G and the synthesis of Structure G-4.

FIG. 7 depicts Chart H and the synthesis of Structure H-9.

FIG. 8 depicts Chart I and the synthesis of Structures I-4 through I-7.

FIGS. 9A-9B depict Chart J and the synthesis of Structures J-3 and J-4.

FIGS. 10A-10B depict Chart K and the synthesis of Structure K-9.

DETAILED DESCRIPTION Definitions

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e., C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups thatare limited to hydrocarbon groups are termed “homoalkyl”. Unlessotherwise specified, each instance of an alkyl group is independentlyoptionally substituted, i.e., unsubstituted (an “unsubstituted alkyl”)or substituted (a “substituted alkyl”) with one or more substituents;e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1substituent. In certain embodiments, the alkyl group is unsubstitutedC₁₋₁₀ alkyl (e.g., —CH₃). In certain embodiments, the alkyl group issubstituted C₁₋₁₀ alkyl. Common alkyl abbreviations include Me (—CH₃),Et (—CH₂CH₃), iPr (—CH(CH₃)₂), nPr (—CH₂CH₂CH₃), n-Bu (—CH₂CH₂CH₂CH₃),or i-Bu (—CH₂CH(CH₃)₂).

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The term “alkenyl” refers to a straight or branched hydrocarbon chaincontaining 2-12 carbon atoms (unless otherwise noted) and having one ormore double bonds. Examples of alkenyl groups include, but are notlimited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups.One of the double bond carbons may optionally be the point of attachmentof the alkenyl substituent.

The term “alkenylene” refers to a divalent alkenyl, e.g. —CH≡CH—,—CH₂-CH≡CH—, and —CH≡CH-CH₂—.

The term “alkynyl” refers to a straight or branched hydrocarbon chaincontaining 2-12 carbon atoms (unless otherwise noted) and characterizedin having one or more triple bonds. Examples of alkynyl groups include,but are not limited to, ethynyl, propargyl, and 3-hexynyl. One of thetriple bond carbons may optionally be the point of attachment of thealkynyl substituent.

The term “alkynylene” refers to a divalent alkynyl, e.g. —CH≡CH—,—CH₂-CH≡CH—, and —CH≡CH—CH₂—.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The terms “cyano” and “nitrile” refer to the radical —CN.

The terms “cycloalkyl”, “heterocycloalkyl” or “heterocyclyl”, bythemselves or in combination with other terms, represent, unlessotherwise stated, cyclic versions of “alkyl” and “heteroalkyl”,respectively. Additionally, for heterocycloalkyl or heterocyclyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, cyclooctanyl, and the like.Examples of heterocycloalkyl and heterocyclyl include, but are notlimited to, 1-1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “heteroalkyl,” as used herein, refers to an alkyl group, asdefined herein, which further comprises 1 or more (e.g., 1, 2, 3, or 4)heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus)within the parent chain, wherein the one or more heteroatoms is insertedbetween adjacent carbon atoms within the parent carbon chain and/or oneor more heteroatoms is inserted between a carbon atom and the parentmolecule, i.e., between the point of attachment. In certain embodiments,a heteroalkyl group refers to a saturated group having from 1 to 10carbon atoms and 1, 2, 3, or 4 heteroatoms (“heteroC₁ ₁₀alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 9carbon atoms and 1, 2, 3, or 4 heteroatoms (“heteroC₁₋₉ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 8carbon atoms and 1, 2, 3, or 4 heteroatoms (“heteroC₁₋₈ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 7carbon atoms and 1, 2, 3, or 4 heteroatoms (“heteroC₁₋₇ alkyl”). In someembodiments, a heteroalkyl group is a group having 1 to 6 carbon atomsand 1, 2, or 3 heteroatoms (“heteroC₁₋₆ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 5 carbon atoms and 1or 2 heteroatoms (“heteroC₁₋₅ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 4 carbon atoms andfor 2 heteroatoms (“heteroC₁₋₄ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1heteroatom (“heteroC₁₋₃ alkyl”). In some embodiments, a heteroalkylgroup is a saturated group having 1 to 2 carbon atoms and 1 heteroatom(“heteroC₁₋₂ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 carbon atom and 1 heteroatom (“heteroC₁alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 2 to 6 carbon atoms and 1 or 2 heteroatoms (“heteroC₂₋₆ alkyl”).Unless otherwise specified, each instance of a heteroalkyl group isindependently unsubstituted (an “unsubstituted heteroalkyl”) orsubstituted (a “substituted heteroalkyl”) with one or more substituents.In certain embodiments, the heteroalkyl group is an unsubstitutedheteroC₁₋₁₀ alkyl. In certain embodiments, the heteroalkyl group is asubstituted heteroC₁₋₁₀ alkyl.

The terms “heterocyclyl” when used in combination with other terms(e.g., heterocyclylalkyl) includes heterocyclyl rings as defined above.Thus, the term “heterocyclylalkyl” is meant to include those radicals inwhich a heterocyclyl group is attached to an alkyl group including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom.

The term “haloalkyl,” as used herein, refers to an alkyl group, asdefined herein, which further comprises 1 or more (e.g., 1, 2, 3, or 4)halogen atoms (e.g., fluorine, chlorine, bromine, or iodine), whereinthe alkyl group is substituted with one or more halogen atoms. Incertain embodiments, a haloalkyl group refers to a saturated grouphaving from 1 to 10 carbon atoms and 1, 2, 3, or 4 halogen atoms (“haloC₁₋₁₀ alkyl”). Additionally, the term “haloalkyl,” is meant to includemonohaloalkyl and polyhaloalkyl. For example, the term “haloalkyl” ismean to include, but not be limited to, trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The terms “haloalkoxy” or “haloalkoxyl” as used herein, refer to analkoxy group, as defined herein, which further comprises 1 or more(e.g., 1, 2, 3, or 4) halogen atoms (e.g., fluorine, chlorine, bromine,or iodine), wherein the alkoxy group is substituted with one or morehalogen atoms.

The term “hydroxy” refers to the radical —OH.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent that can be a single ring or multiplerings (preferably from 1 to 3 rings), which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl, aralkyl, heteroaralkyl) includesboth aryl and heteroaryl rings as defined above. Thus, the terms“arylalkyl”, “aralkyl” and “heteroaralkyl” are meant to include thoseradicals in which an aryl or heteroaryl group is attached to an alkylgroup (e.g., benzyl, phenethyl, pyridylmethyl and the like) includingthose alkyl groups in which a carbon atom (e.g., a methylene group) hasbeen replaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

The term “nitro” refers to the radical —NO₂.

“Protecting group,” as used herein refers to a portion of a substratethat is substantially stable under a particular reaction condition, butwhich is cleaved from the substrate under a different reactioncondition. A protecting group can also be selected such that itparticipates in the direct oxidation of the aromatic ring component ofthe compounds of the invention. For examples of useful protectinggroups, see, for example, Greene et al., Protective Groups in OrganicSynthesis, John Wiley & Sons, New York, 1991.

Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroarylgroups, as defined herein, are optionally substituted (e.g.,“substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted”alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or“unsubstituted” cycloalkyl, “substituted” or “unsubstituted”heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or“unsubstituted” heteroaryl group). In general, the term “substituted”,whether preceded by the term “optionally” or not, means that at leastone hydrogen present on a group (e.g., a carbon or nitrogen atom) isreplaced with a permissible substituent, e.g., a substituent which uponsubstitution results in a stable compound, e.g., a compound which doesnot spontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction. Unless otherwise indicated,a “substituted” group has a substituent at one or more substitutablepositions of the group, and when more than one position in any givenstructure is substituted, the substituent is either the same ordifferent at each position. The term “substituted” is contemplated toinclude substitution with all permissible substituents of organiccompounds described herein that results in the formation of a stablecompound. For purposes of this invention, heteroatoms such as nitrogenmay have hydrogen substituents and/or any suitable substituent asdescribed herein which satisfy the valencies of the heteroatoms andresults in the formation of a stable moiety.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂,—N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa),—SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂, —CO₂R^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa),—NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR^(aa), —OSO₂R^(aa),—S (O)R^(aa) —S(═O)R^(aa), —OS (═O)R^(aa), —Si(R^(aa))₃, —OSi(R^(aa))₃—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa), —SC(═S)SR^(aa),—SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa), —SC(═O)R^(aa),—P(═O)₂R^(aa), —OP(═O)₂R^(aa), —P(═O)(R^(aa))₂, —OP(═O)(R^(aa))₂,—OP(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, —OP(═O)₂N(R^(bb))₂,—P(═O)(NR^(bb))₂, —OP(═O)(NR^(bb))₂, —NR^(bb)P(═O)(OR^(cc))₂,—NR^(bb)P(═O)(NR^(bb))₂, —P(R^(cc))₂, —P(R^(cc))₃, —OP(R^(cc))₂,—OP(R^(cc))₃, —B(R^(aa))₂, —B(OR^(cc))₂, —BR^(aa)(OR^(cc)), C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ cycloalkyl, 3-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, whereineach alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd)groups;

-   -   each instance of R^(aa) is, independently, selected from C₁₋₁₀        alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀        cycloalkyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14        membered heteroaryl, or two R^(aa) groups are joined to form a        3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,        wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl,        aryl, and heteroaryl is independently substituted with 0, 1, 2,        3, 4, or 5 R^(dd) groups;    -   each instance of R^(bb) is, independently, selected from        hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),        —C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(cc))OR^(aa),        —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),        —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),        —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂,        —P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ cycloalkyl, 3-14 membered        heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two        R^(bb) groups are joined to form a 3-14 membered heterocyclyl or        5-14 membered heteroaryl ring, wherein each alkyl, alkenyl,        alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is        independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd)        groups;    -   each instance of R^(cc) is, independently, selected from        hydrogen, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀        alkynyl, C₃₋₁₀ cycloalkyl, 3-14 membered heterocyclyl, C₆₋₁₄        aryl, and 5-14 membered heteroaryl, or two R^(cc) groups are        joined to form a 3-14 membered heterocyclyl or 5-14 membered        heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,        cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently        substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;    -   each instance of R^(dd) is, independently, selected from        halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee),        —ON(R^(ff))₂, —N(R^(ff))₂, —N(R^(ff))₃ ⁺X⁻, —N(OR^(ee))R^(ff),        —SH, —SR^(ee), —SSR^(ee), —C(═O)R^(ee), —CO₂H, —CO₂R^(ee),        —OC(═O)R^(ee), —OCO₂R^(ee), —C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂,        —NR^(ff)C(═O)R^(ee), —NR^(ff)CO₂R^(ee), —NR^(ff)C(═O)N(R^(ff))₂,        —C(═NR^(ff))OR^(ee), —OC(═NR^(ff))R^(ee), —OC(═NR^(ff))OR^(ee),        —C(═NR^(ff))N(R^(ff))₂, —OC(═NR^(ff))N(R^(ff))₂,        —NR^(ff)C(═NR^(ff))N(R^(ff))₂, —NR^(ff)SO₂R^(ee),        —SO₂N(R^(ff))₂, —SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee),        —S(═O)R^(ee), —Si(R^(ee))₃, —OSi(R^(ee))₃, —C(═S)N(R^(ff))₂,        —C(═O)SR^(ee), —C(═S)SR^(ee), —SC(═S)SR^(ee), —P(═O)₂R^(ee),        —P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂, —OP(═O)(OR^(ee))₂, C₁₋₆        alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀        cycloalkyl, 3-10 membered heterocyclyl, C₆₋₁₀ aryl, 5-10        membered heteroaryl, wherein each alkyl, alkenyl, alkynyl,        cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently        substituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups, or two        geminal R^(dd) substituents can be joined to form ═O or ═S;    -   each instance of R^(ee) is, independently, selected from C₁₋₆        alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀        cycloalkyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, and 3-10        membered heteroaryl, wherein each alkyl, alkenyl, alkynyl,        cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently        substituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups;    -   each instance of R^(ff) is, independently, selected from        hydrogen, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆        alkynyl, C₃₋₁₀ cycloalkyl, 3-10 membered heterocyclyl, C₆₋₁₀        aryl and 5-10 membered heteroaryl, or two R^(gg) groups are        joined to form a 3-14 membered heterocyclyl or 5-14 membered        heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,        cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently        substituted with 0, 1,2,3,4, or 5 R^(gg) groups; and    -   each instance of R^(gg) is, independently, halogen, —CN, —NO₂,        —N₃, —SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆        alkyl)₂, N(C₁₋₆ alkyl)₃ ⁺X⁻, —NH(C₁₋₆ alkyl)₂ ⁺X⁻, —NH₂(C₁₋₆        alkyl) ⁺X³¹ , —NH₃ ⁺X⁻, —N(OC₁₋₆ alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆        alkyl), —NH(OH), —SH, —SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl), —C(═O)(C₁₋₆        alkyl), —CO₂H, —CO₂(C₁₋₆ alkyl), —OC(═O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆        alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆ alkyl)₂, —OC(═O)NH(C₁₋₆ alkyl),        —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆ alkyl)C(═O)(C₁₋₆ alkyl),        —NHCO₂(C₁₋₆ alkyl), —NHC(═O)N(C₁₋₆ alkyl)₂, —NHC(═O)NH(C₁₋₆        alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆ alkyl),        —OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆        alkyl), —C(═NH)NH₂, —OC(═NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆        alkyl), —OC(NH)NH₂, —NHC(NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂,        —NHSO₂(C₁₋₆ alkyl), —SO₂N(C₁₋₆ alkyl)₂, —SO₂NH(C₁₋₆ alkyl),        —SO₂NH₂, —SO₂C₁₋₆ alkyl, —SO₂OC₁₋₆ alkyl, —OSO₂C₁₋₆ alkyl,        —SOC₁₋₆ alkyl, —Si(C₁₋₆ alkyl)₃, —OSi(C₁₋₆ alkyl)₃ —C(═S)N(C₁₋₆        alkyl)₂, —C(═S)NH(C₁₋₆ alkyl), —C(═S)NH₂, —C(═O)S(C₁₋₆ alkyl),        —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)₂(C₁₋₆ alkyl),        —P(═O)(C₁₋₆ alkyl)₂, —OP(═O)(C₁ ₆ alkyl)₂, —OP(═O)(OC₁ ₆        alkyl)₂, C₁₋₆ alkyl, C₁ ₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆        alkynyl, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, 3-10 membered        heterocyclyl, 5-10 membered heteroaryl; or two geminal R^(gg)        substituents can be joined to form ═O or ═S; wherein X⁻ is a        counterion.        These and other exemplary substituents are described in more        detail in the Detailed Description, Examples, and claims. The        invention is not intended to be limited in any manner by the        above exemplary listing of substituents.

Compounds

Described herein are compounds that inhibit Factor XIa or kallikrein,for example the compounds described herein (e.g., a compound of formula(I)-(VII)).

Exemplary compounds include, but are not limited to the compoundsdescribed in Table 1 below:

TABLE 1 Exemplary compounds of the present invention. ID# Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

31

32

33

34

35

36

37

38

39

40

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

83

84

86

88

89

91

92

93

95

96

97

98

99

100

101

102

103

104

105

107

108

109

110

111

112

113

114

117

118

119

120

123

124

126

127

128

130

131

132

133

134

135

136

137

138

139

140

144

145

147

148

149

150

151

152

153

154

155

156

157

158

159

160

166

168

169

170

171

175

180

190

191

192

193

194

195

196

197

198

199

200

In some embodiments, a compound described herein is formed into a salt.A compound described herein can be administered as a free acid, azwitterion or as a salt. A salt can also be formed between a cation anda negatively charged substituent on a compound described herein.Suitable cationic counterions include sodium ions, potassium ions,magnesium ions, calcium ion, and ammonium ions (e.g., a tetraalkylammonium cation such as tetramethylammonium ion). In compounds includinga positively charged substituent or a basic substituent, a salt can beformed between an anion and a positively charged substituent (e.g.,amino group) or basic substituent (e.g., pyridyl) on a compounddescribed herein. Suitable anions include chloride, bromide, iodide,sulfate, nitrate, phosphate, citrate, methanesulfonate,trifluoroacetate, and acetate.

Pharmaceutically acceptable salts of the compounds described herein(e.g., a compound of formula (I)-(VII)) also include those derived frompharmaceutically acceptable inorganic and organic acids and bases.Examples of suitable acid salts include acetate, adipate, alginate,aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate,camphorate, camphorsulfonate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate,pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,propionate, salicylate, succinate, sulfate, tartrate, thiocyanate,tosylate, trifluoroacetate, and undecanoate.

Salts derived from appropriate bases include alkali metal (e.g.,sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄⁺ salts. This invention also envisions the quaternization of any basicnitrogen-containing groups of the compounds disclosed herein. Water oroil-soluble or dispersible products may be obtained by suchquaternization.

As used herein, the compounds of this invention, including the compoundsof formula (I)-(VII), are defined to include pharmaceutically acceptablederivatives or prodrugs thereof. A “pharmaceutically acceptablederivative or prodrug” means any pharmaceutically acceptable salt,ester, salt of an ester, or other derivative of a compound of thisinvention which, upon administration to a recipient, is capable ofproviding (directly or indirectly) a compound of this invention.Particularly favored derivatives and prodrugs are those that increasethe bioavailability of the compounds of this invention when suchcompounds are administered to a mammal (e.g., by allowing an orallyadministered compound to be more readily absorbed into the blood), orwhich enhance delivery of the parent compound to a biologicalcompartment (e.g., the brain or lymphatic system) relative to the parentspecies. Preferred prodrugs include derivatives where a group whichenhances aqueous solubility or active transport through the gut membraneis appended to the structure of formulae described herein.

Any formula or a compound described herein is also intended to representunlabeled forms as well as isotopically labeled forms of the compounds,isotopically labeled compounds have structures depicted by the formulasgiven herein except that one or more atoms are replaced by an atomhaving a selected atomic mass or mass number. Examples of isotopes thatcan be incorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine,such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F ⁵¹P, ³²P, ³⁵S, ³⁶Cl, , ¹²⁵Irespectively. The invention includes various isotopically labeledcompounds as defined herein, for example, those into which radioactiveisotopes, such as ³H, ¹³C, and ¹⁴C are present. Such isotopicallylabelled compounds are useful in metabolic studies (with ¹⁴C), reactionkinetic studies (with, for example ¹H or ³H), detection or imagingtechniques, such as positron emission tomography (PET) or single-photonemission computed tomography (SPECT) including drug or substrate tissuedistribution assays, or in radioactive treatment of patients. Inparticular, an ¹⁸F or labeled compound may be particularly desirable forPET or SPECT studies, isotopically labeled compounds of this inventionand prodrugs thereof can generally be prepared by carrying out theprocedures disclosed in the schemes or in the examples and preparationsdescribed below by substituting a readily available isotopically labeledreagent for a non-isotopically labeled reagent.

Further, substitution with heavier isotopes, particularly deuterium(i.e., ²H or D) may afford certain therapeutic advantages resulting fromgreater metabolic stability, for example increased in vivo half-life orreduced dosage requirements or an improvement in therapeutic index. Itis understood that deuterium in this context is regarded as asubstituent of a compound of a formula described herein. Theconcentration of such a heavier isotope, specifically deuterium, may bedefined by the isotopic enrichment factor. The term “isotopic enrichmentfactor” as used herein means the ratio between the isotopic abundanceand the natural abundance of a specified isotope If a substituent m acompound of this invention is denoted deuterium, such compound has anisotopic enrichment factor for each designated deuterium atom of atleast 3500 (52.5% deuterium incorporation at each designated deuteriumatom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5%deuterium incorporation), at least 5000 (75% deuterium incorporation),at least 5500 (82.5% deuterium incorporation), at least 6000 (90%deuterium incorporation), at least 6333.3 (95% deuterium incorporation),at least 6466.7 (97% deuterium incorporation), at least 6600 (99%deuterium incorporation), or at least 8633.3 (99.5% deuteriumincorporation).

Isotopically-labelled compounds described herein can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described in the accompanying Examplesand Preparations using an appropriate isotopically-labeled reagents inplace of the non-labeled reagent previously employed. Pharmaceuticallyacceptable solvates in accordance with the invention include thosewherein the solvent of crystallization may be isotopically substituted,e.g, D₂O, D₂-acetone, D₂-DMSO.

Any asymmetric atom (e.g., carbon or the like) of the compound(s) of thepresent invention can be present in racemic or enantiomericallyenriched, for example the (R)- (S)- or (RS)- configuration, in certainembodiments, each asymmetric atom has at least 50% enantiomeric excess,at least 60% enantiomeric excess, at least 70% enantiomeric excess, atleast 80% enantiomeric excess, at least 90% enantiomeric excess, atleast 95% enantiomeric excess, or at least 99% enantiomeric excess inthe (R)- or (S)- configuration. Substituents at atoms with unsaturatedbonds may, if possible, be present in cis-(Z)- or trans- (E)- forrnAccordingly, as used herein a compound of the present invention can bein the form of one of the possible isomers, rotamers, atropisomers,tautoroers or mixtures thereof, for exampie, as substantially puregeometric (cis or trans) isomers, diastereorners, optical isomers(antipodes), racemates or mixtures thereof. Any resulting mixtures ofisomers can be separated on the basis of the physicochemical differencesof the constituents, into the pure or substantially pure geometric oroptical isomers, diastereomers, racemates, for example, bychromatography or fractional crystallization.

Any resulting racemates of final products or intermediates can beresolved into the optical antipodes by known methods, e.g., byseparation of the diastereomeric salts thereof, obtained with anoptically active acid or base, and liberating the optically activeacidic or basic compound. In particular, a basic moiety may thus beemployed to resolve the compounds of the present invention into theiroptical antipodes, e.g., by fractional crystallization of a salt formedwith an optically active acid, e.g., tartaric acid, dibenzoyl tartaricacid, diacetyl tartaric acid, (+) —O,O′-Di-p-toluoyl-D-tartaric acid,mandelic acid, malic acid or camphor-10-sulfonic acid. Racemic productscan also be resolved by chiral chromatography, e.g., high pressureliquid chromatography (HPLC) using a chiral adsorbent.

The compounds described herein (e.g., a compound of formula (I)-(VII))may also be represented in multiple tautomeric forms, for example, acompound of formula (I), (II), (III), (IV), (V), (VI), or (VII). In suchinstances, the invention expressly includes all tautomeric forms of thecompounds described herein. All crystal forms of the compounds describedherein are expressly included in this invention.

A compound described herein (e.g., a compound of formula (I)-(VII)) canbe evaluated for its ability to modulate (e.g., inhibit) Factor XIa orkallikrein.

Methods of Synthesizing Compounds

The compounds described herein can be synthesized by conventionalmethods using commercially available starting materials and reagents.For example, compounds can be synthesized utilizing the methods setforth in U.S. Pat. No. 7,501,404, or as described in the methodsdescribed herein.

Methods of Treatment

The compounds described herein (e.g., compounds of formula (I)-(VII))can inhibit Factor XIa or kallikrein. In some embodiments, a compounddescribed herein can inhibit both Factor XIa and kallikrein. As aresult, these compounds can be useful in the treatment or prevention ofa disorder described herein. Exemplary disorders include thromboticevents associated with coronary artery and cerebrovascular disease,venous or arterial thrombosis, coagulation syndromes, ischemia andangina (stable and unstable), deep vein thrombosis (DVT), disseminatedintravascular coagulopathy, Kasabach-Merritt syndrome, pulmonaryembolism, myocardial infarction, cerebral infarction, cerebralthrombosis, transient ischemic attacks, atrial fibrillation, cerebralembolism, thromboembolic complications of surgery (such as hip or kneereplacement, introduction of artificial heart valves and endarterectomy)and peripheral arterial occlusion and may also be useful in treating orpreventing myocardial infarction, stroke, angina and other consequencesof atherosclerotic plaque rupture. The compounds of the inventionpossessing Factor XIa or kallikrein inhibition activity may also beuseful in preventing thrombosis in cancer patients and to preventthromboembolic events at or following tissue plasminogen activator-basedor mechanical restoration of blood vessel patency. The compounds of theinvention possessing Factor XIa or kallikrein inhibition activity mayalso be useful as inhibitors of blood coagulation such as during thepreparation, storage and fractionation of whole blood.

Factor XIa inhibition, according to the present invention, can be a moreeffective and safer method of inhibiting thrombosis compared toinhibiting other coagulation serine proteases such as thrombin or FactorXa. Administration of a small molecule Factor XIa inhibitor should havethe effect of inhibiting thrombin generation and clot formation with noor substantially no effect on bleeding times and little or no impairmentof haemostasis. These results differ substantially from that of other“direct acting” coagulation protease inhibitors (e.g., active-siteinhibitors of thrombin and Factor Xa), which demonstrate prolongation ofbleeding time and less separation between antithrombotic efficacy andbleeding time prolongation. A preferred method according to theinvention comprises administering to a mammal a pharmaceuticalcomposition containing at least one compound of the invention.

The compounds described herein (e.g., any of a compound of formula(I)-(VII)) can inhibit kallikrein, for example, a compound of formula(I), (II), (III), (IV), (V), (VI), or (VII). As a result, thesecompounds can be useful in the treatment or prevention of diseasesinvolved in inflammation, such as edema (e.g., cerebral edema, macularedema, and angioedema (e.g., hereditary angioedema)). In someembodiments, the compounds of the invention can be useful in thetreatment or prevention of hereditary angioedema. The compoundsdescribed herein (e.g., compounds of formula (I)-(VII)) can also beuseful in the treatment of e.g., stroke, ischemia, and perioperativeblood loss for example, a compound of formula (I), (II), (III), (IV),(V), (VI), or (VII).

The methods of the present invention are useful for treating orpreventing those conditions which involve the action of Factor XIa orkallikrein. Accordingly, the methods of the present invention are usefulin treating consequences of atherosclerotic plaque rupture includingcardiovascular diseases associated with the activation of thecoagulation cascade in thrombotic or thrombophilic states. As usedherein, the terms “treating” or “treatment” encompass responsive orprophylaxis measures, e.g., measures designed to inhibit or delay theonset of the disease, achieve a full or partial reduction of thesymptoms or disease state, or to alleviate, lessen, or cure the diseaseor disorder or its symptoms.

More particularly, the methods of the present invention may be used totreat acute coronary syndromes such as coronary artery disease,myocardial infarction, unstable angina (including crescendo angina),ischemia (e.g., ischemia resulting from vascular occlusion), andcerebral infarction. The methods of the present invention further may beuseful in treating stroke and related cerebral vascular diseases(including cerebrovascular accident, vascular dementia, and transientischemic attack); venous thrombosis and thrombo-embolism, such as deepvein thrombosis (DVT) and pulmonary embolism; thrombosis associated withatrial fibrillation, ventricular enlargement, dilated cardiac myopathy,or heart failure; peripheral arterial disease and intermittentclaudication; the formation of atherosclerotic plaques and transplantatherosclerosis; restenosis following arterial injury inducedendogenously (by rupture of an atherosclerotic plaque), or exogenously(by invasive cardiological procedures such as vessel wall injuryresulting from angioplasty); disseminated intravascular coagulopathy,Kasabach-Merritt syndrome, cerebral thrombosis, and cerebral embolism.

Additionally, the methods of the present invention may be useful intreating thrombo-embolic consequences or complications associated withcancer; surgery (such as hip replacement, endarterectomy, introductionof artificial heart valves, vascular grafts, mechanical organs, andimplantation or transplantation of organ, tissue or cells); medications(such as tissue plasminogen activator or similar agents and surgicalrestoration of blood vessel patency) in patients suffering myocardialinfarction, stroke, pulmonary embolism and like conditions; medications(such as oral contraceptives, hormone replacement, and heparin, e.g.,for treating heparin-induced thrombocytopenia); sepsis (such as sepsisrelated to disseminated intravascular coagulation); and pregnancy orchildbirth. The methods of the present invention may be used to treatthrombosis due to confinement (i.e. immobilization, hospitalization, bedrest, limb immobilization, e.g., with immobilizing casts, etc.).

The methods of the present invention may also be used to maintain bloodvessel patency, for example, in patients undergoing transluminalcoronary angioplasty, or in connection with vascular surgery such asbypass grafting, arterial reconstruction, atherectomy, vascular grafts,stent patency, and organ, tissue or cell implantation andtransplantation. The inventive methods may be used to inhibit bloodcoagulation in connection with the preparation, storage, fractionation,or use of whole blood. For example, the inventive methods may be used inmaintaining whole and fractionated blood in the fluid phase such asrequired for analytical and biological testing, e.g., for ex vivoplatelet and other cell function studies, bioanalytical procedures, andquantitation of blood-containing components, or for maintainingextracorpeal blood circuits, as in dialysis or surgery (e.g., coronaryartery bypass surgery).

In addition, the methods of the present invention may be useful intreating and preventing the prothrombotic complications of cancer. Themethods may be useful in treating tumor growth, as an adjunct tochemotherapy, for preventing angiogenesis, and for treating cancer, moreparticularly, cancer of the lung, prostate, colon, breast, ovaries, andbone.

Ischemia

“Ischemia” or an “ischemic event” is a vascular disease generallyinvolving vascular occlusion or a restriction in blood supply totissues. Ischemia can cause a shortage of oxygen and glucose needed forcellular metabolism. Ischemia is generally caused by problematic bloodvessels that result in damage or dysfunction of tissue. Ischemia canalso refer to a local loss in blood or oxygen in a given part of thebody resulting from congestion (e.g., vasoconstriction, thrombosis, orembolism). Causes include embolism, thrombosis of an atherosclerosisartery, trauma, venous problems, aneurysm, heart conditions (e.g.,myocardial infarction, mitral valve disease, chronic arterialfibrillation, cardiomyopathies, and prosthesis), trauma or traumaticinjury (e.g., to an extremity producing partial or total vesselocclusion), thoracic outlet syndrome, atherosclerosis, hypoglycemia,tachycardia, hypotension, outside compression of a blood vessel (e.g.,by a tumor), sickle cell disease, localized extreme cold (e.g., byfrostbite), tourniquet application, glutamate receptor stimulation,arteriovenous malformations, rupture of significant blood vesselssupplying a tissue or organ, and anemia.

A transient ischemic event generally refers to a transient (e.g.,short-lived) episode of neurologic dysfunction caused by loss of bloodflow (e.g., in the focal brain, spinal cord, or retinal) without acuteinfarction (e.g., tissue death). In some embodiments, the transientischemic event lasts for less than 72 hours, 48 hours, 24 hours, 12hours, 10 hours, 8 hours, 4 hours, 2 hours, 1 hour, 45 minutes, 30minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3minutes, 2 minutes, or 1 minute.

Angioedema

Angioedema is the rapid swelling of the dermis, subcutaneous tissue,mucosa, and submucosal tissues. Angioedema is typically classified aseither hereditary or acquired.

“Acquired angioedema” can be immunologic, non-immunologic, oridiopathic; caused by e.g., allergy, as a side effect of medications,e.g., ACE inhibitor medications.

“Hereditary angioedema” or “HAE” refers to a genetic disorder thatresults in acute periods of edema (e.g., swelling) that may occur innearly all parts of the body, including the face, limbs, neck, throat,larynx, extremities, gastrointestinal tract, and genitalia. Attacks ofHAE can often be life-threatening, with severity depending on the areaaffected, e.g., abdominal attacks may result in intenstinal obstruction,while swelling of the larynx and upper airway can lead to asphyxiation.Pathogenesis of hereditary angioedema may be related to unopposedactivation of the contact pathway by the initial generation ofkallikrein or clotting factors (e.g., Factor XII).

Signs and symptoms include swelling, e.g., of the skill of the face,mucosa of the mouth or throat, and tongue. Itchiness, pain, decreasedsensation in the affected areas, urticaria (i.e., hives), or stridor ofthe airway may also be a sign of angioedema. However, there can be noassociated itch, or urticaria, e.g., in hereditary angioedema. HAEsubjects can experience abdominal pain (e.g., abdominal pain lasting oneto five days, abdominal attacks increasing a subject's white blood cellcount), vomiting, weakness, watery diarrhea, or rash.

Bradykinin plays an important role in angioedema, particularlyhereditary angioedema. Bradykinin is released by various cell types inresponse to numerous different stimuli and is a pain mediator.Interfering with bradykinin production or degradation can lead toangioedema. In hereditary angioedema, continuous production of enzymekallikrein can facilitate bradykinin formation. Inhibition of kallikreincan interfere with bradykinin production; and treat or preventangioedema.

Pharmaceutical Compositions

The compositions described herein include the compound described herein(e.g., a compound of formula (I)-(VII), e.g., a compound of formula (I),(II), (III), (IV), (V), (VI), or (VII)), as well as additionaltherapeutic agents, if present, in amounts effective for achieving thetreatment of a disease or disease symptoms (e.g., such as a diseaseassociated with Factor XIa or kallikrein).

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions provided herewith include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, orchemically modified derivatives such as hydroxyalkylcyclodextrins,including 2 - and 3-hydroxypropyl-β-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery ofcompounds of the formulae described herein.

Routes of Administration

The pharmaceutical compositions provided herewith may be administeredorally, rectally, or parenterally (e.g., intravenous infusion,intravenous bolus injection, inhalation, implantation). The termparenteral as used herein includes subcutaneous, intracutaneous,intravenous (e.g., intravenous infusion, intravenous bolus injection),intranasal, inhalation, pulmonary, transdermal, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional and intracranial injection or other infusion techniques.The pharmaceutical compositions provided herewith may contain anyconventional non-toxic pharmaceutically-acceptable carriers, adjuvantsor vehicles. In some cases, the pH of the formulation may be adjustedwith pharmaceutically acceptable acids, bases or buffers to enhance thestability of the formulated compound or its delivery form.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous solution or suspension. This suspension may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents (such as, for example, Tween 80) and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are mannitol,water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose, any bland fixed oil may beemployed including synthetic mono- or diglycerides. Fatty acids, such asoleic acid and its glyceride derivatives are useful in the preparationof injectables, as are natural pharmaceutically-acceptable oils, such asolive oil or castor oil, especially in their polyoxyethylated versions.These oil solutions or suspensions may also contain a long-chain alcoholdiluent or dispersant, or carboxymethyl cellulose or similar dispersingagents which are commonly used in the formulation of pharmaceuticallyacceptable dosage forms such as emulsions and or suspensions. Othercommonly used surfactants such as Tweens or Spans or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms may also be used for the purposes of formulation.

The pharmaceutical compositions provided herewith may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase is combined with emulsifying or suspending agents. Ifdesired, certain sweetening or flavoring or coloring or taste maskingagents may be added.

The compounds described herein can, for example, be administered byinjection, intravenously (e.g., intravenous infusion, intravenous bolusinjection), intraarterially, subdermally, intraperitoneally,intramuscularly, or subcutaneously; or orally, buccally, nasally,transmucosally, topically with a dosage ranging from about 0.5 to about100 mg/kg of body weight, alternatively dosages between 1 mg and 1000mg/dose, every 4 to 120 hours, or according to the requirements of theparticular drug. The methods herein contemplate administration of aneffective amount of compound or compound composition to achieve thedesired or stated effect. Typically, the pharmaceutical compositionsprovided herewith will be administered from about 1 to about 6 times perday (e.g., by intravenous bolus injection) or alternatively, as acontinuous infusion. Such administration can be used as a chronic oracute therapy. The amount of active ingredient that may be combined withthe carrier materials to produce a single dosage form will varydepending upon the host treated and the particular mode ofadministration. A typical preparation will contain from about 5% toabout 95% active compound (w/w). Alternatively, such preparationscontain from about 20% to about 80% active compound.

Combinations

In carrying out the methods of the present invention, it may be desiredto administer the compounds of the invention (Factor XIa or kallikreininhibitors) in combination with each other and one or more other agentsfor achieving a therapeutic benefit such as antithrombotic oranticoagulant agents, anti-hypertensive agents, anti-ischemic agents,anti-arrhythmic agents, platelet function inhibitors, and so forth. Forexample, the methods of the present invention may be carried out byadministering the small molecule Factor XIa or kallikrein inhibitors incombination with a small molecule Factor XIa or kallikrein inhibitor.More particularly, the inventive methods may be carried out byadministering the small molecule Factor XIa or kallikrein inhibitors incombination with aspirin, clopidogrel, ticlopidine or CS-747, warfarin,low molecular weight heparins (such as LOVENOX), GPIIb/GPIIIa blockers,PAI-1 inhibitors such as XR-330 and T-686, P2Y1 and P2Y12 receptorantagonists; thromboxane receptor antagonists (such as ifetroban),prostacyclin mimetics, thromboxane A synthetase inhibitors (such aspicotamide), serotonin-2-receptor antagonists (such as ketanserin);compounds that inhibit other coagulation factors such as FVII, FVIII,FIX, FX, prothrombin, TAFI, and fibrinogen, or other compounds thatinhibit FXI or kallikrein; fibrinolytics such as TPA, streptokinase,PAI-1 inhibitors, and inhibitors of α-2-antiplasmin such asanti-α-2-antiplasmin antibody fibrinogen receptor antagonists,inhibitors of α-1-antitrypsin, hypolipidemic agents, such as HMG-CoAreductase inhibitors (e.g., pravastatin, simvastatin, atorvastatin,fluvastatin, cerivastatin, AZ4522, and itavastatin), and microsomaltriglyceride transport protein inhibitors (such as disclosed in U.S.Pat. Nos. 5,739,135, 5,712,279 and 5,760,246); antihypertensive agentssuch as angiotensin-converting enzyme inhibitors (e.g., captopril,lisinopril or fosinopril); angiotensin-II receptor antagonists (e.g.,irbesartan, losartan or valsartan); ACE/NEP inhibitors (e.g.,omapatrilat and gemopatrilat); or β-blockers (such as propranolol,nadolol and carvedilol). The inventive methods may be carried out byadministering the small molecule Factor XIa or kallikrein inhibitors incombination with anti-arrhythmic agents such as for atrial fibrillation,for example, amiodarone or dofetilide.

In carrying out the methods of the present invention, it may be desiredto administer the compounds of the invention (Factor XIa or kallikreininhibitors) in combination with agents that increase the levels of cAMPor cGMP in cells for a therapeutic benefit. For example, the compoundsof the invention may have advantageous effects when used in combinationwith phosphodiesterase inhibitors, including PDE1 inhibitors (such asthose described in Journal of Medicinal Chemistry, Vol. 40, pp.2196-2210 [1997]), PDE2 inhibitors, PDE3 inhibitors (such as revizinone,pimobendan, or olprinone), PDE4 inhibitors (such as rolipram,cilomilast, or piclamilast), PDE7 inhibitors, or other PDE inhibitorssuch as dipyridamole, cilostazol, sildenafil, denbutyline, theophylline(1,2-dimethylxanthine), ARIFLOT.TM. (i.e.,cis-4-cyano-4-[3-(cyclopentylox-y)-4-methoxyphenyl]cyclohexane-1-carboxyl-ic acid), arofyline, roflumilast, C-11294A, CDC-801, BAY-19-8004,cipamfylline, SCH351591, YM-976, PD-189659, mesiopram, pumafentrine,CDC-998, IC-485, and KW-4490.

The inventive methods may be carried out by administering the compoundsof the invention in combination with prothrombolytic agents, such astissue plasminogen activator (natural or recombinant), streptokinase,reteplase, activase, lanoteplase, urokinase, prourokinase, anisolatedstreptokinase plasminogen activator complex (ASPAC), animal salivarygland plasminogen activators, and the like.

The inventive methods may be carried out by administering the compoundsof the invention in combination with β-adrenergic agonists such asalbuterol, terbutaline, formoterol, salmeterol, bitolterol, pilbuterol,or fenoterol; anticholinergics such as ipratropium bromide;anti-inflammatory cortiocosteroids such as beclomethasone,triamcinolone, budesonide, fluticasone, flunisolide or dexamethasone;and anti-inflammatory agents such as cromolyn, nedocromil, theophylline,zileuton, zafirlukast, monteleukast and pranleukast.

Small molecule Factor XIa or kallikrein inhibitors may actsynergistically with one or more of the above agents. Thus, reduceddoses of thrombolytic agent(s) may be used, therefore obtaining thebenefits of administering these compounds while minimizing potentialhemorrhagic and other side effects.

Course of Treatment

The compositions described herein include a therapeutically effectiveamount of a compound of the invention (e.g., a Factor XIa or kallikreininhibitor) in combination and one or more other agents (e.g., anadditional therapeutic agent) such as antithrombotic or anticoagulantagents, anti-hypertensive agents, anti-ischemic agents, anti-arrhythmicagents, platelet function inhibitors, and so forth for achieving atherapeutic benefit.

In some embodiments, the additional therapeutic agent is administeredfollowing administration of the compound of the invention (e.g., aFactor XIa or kallikrein inhibitor). In some embodiments, the additionaltherapeutic agent is administered 15 minutes, 30 minutes, 1 hour, 2hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 18hours, 24 hours, 48 hours, 72 hours or longer after administration ofthe compound of the invention (e.g., a Factor XIa or kallikreininhibitor). In some embodiments, the additional therapeutic agent isadministered (e.g., orally) after discharge from a medical facility(e.g., a hospital).

In some embodiments, the compound of the invention (e.g., a Factor XIaor kallikrein inhibitor) and additional therapeutic agent areco-formulated into a single composition or dosage. In some embodiments,the compound of the invention (e.g., a Factor XIa or kallikreininhibitor) and additional therapeutic agent are administered separately.In some embodiments, the compound of the invention (e.g., a Factor XIaor kallikrein inhibitor) and additional therapeutic agent areadministered sequentially. In some embodiments, the compound of theinvention (e.g., a Factor XIa or kallikrein inhibitor) and additionaltherapeutic agent are administered separately and sequentially. Ingeneral, at least one of the compound of the invention (e.g., a FactorXIa or kallikrein inhibitor) and additional therapeutic agent isadministered parenterally (e.g., intranasally, intramuscularly buccally,inhalation, implantation, transdermal, intravenously (e.g., intravenousinfusion, intravenous bolus injection), subcutaneous, intracutaneous,intranasal, pulmonary, transdermal, intraarticular, intraarterial,intrasynovial, intrasternal, intrathecal, intralesional and intracranialinjection or other infusion techniques); orally; or rectally, forexample, intramuscular injection or intravenously (e.g., intravenousinfusion, intravenous bolus injection)). In some embodiments, compoundof the invention is administered parenterally (e.g., intranasally,buccally, intravenously (e.g., intravenous infusion, intravenous bolusinjection) or intramuscularly). In some embodiments, additionaltherapeutic agent is administered orally. In some embodiments, thecompound of the invention (e.g., a Factor XIa or kallikrein inhibitor)is administered parenterally (e.g., intranasally, buccally,intravenously (e.g., intravenous infusion, intravenous bolus injection)or intramuscularly) and the additional therapeutic agent is administeredorally.

In some embodiments, the compound of the invention (e.g., a Factor XIaor kallikrein inhibitor) may be administered once or several times aday. A duration of treatment may follow, for example, once per day for aperiod of about 1, 2, 3, 4, 5, 6, 7 days or more. In some embodiments,the treatment is chronic (e.g., for a lifetime). In some embodiments,the treatment is administered orally. In some embodiments, either asingle dose in the form of an individual dosage unit or several smallerdosage units or by multiple administrations of subdivided dosages atcertain intervals is administered. For instance, a dosage unit can beadministered from about 0 hours to about 1 hr, about 1 hr to about 24hr, about 1 to about 72 hours, about 1 to about 120 hours, or about 24hours to at least about 120 hours post injury. Alternatively, the dosageunit can be administered from about 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 40, 48,72, 96, 120 hours or longer post injury. Subsequent dosage units can beadministered any time following the initial administration such that atherapeutic effect is achieved. In some embodiments, the initial dose isadministered orally. In some embodiments, doses subsequent to theinitial dose are administered parenterally (e.g., intranasally,intramuscularly buccally, inhalation, implantation, transdermal,intravenously (e.g., intravenous infusion, intravenous bolus injection),subcutaneous, intracutaneous, intranasal, pulmonary, transdermal,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional and intracranial injection or other infusion techniques);orally; or rectally.

Where a subject undergoing therapy exhibits a partial response, or arelapse following completion of the first cycle of the therapy,subsequent courses of therapy may be needed to achieve a partial orcomplete therapeutic response (e.g., chronic treatment, e.g., for alifetime).

In some embodiments, the compound of the invention (e.g., a Factor XIaor kallikrein inhibitor) is administered intravenously, e.g., as anintravenous infusion or intravenous bolus injection, for about 5 minutesto about 1 week; about 30 minutes to about 24 hours, about 1 hour toabout 12 hours, about 2 hours to about 12 hours, about 4 hours to about12 hours, about 6 hours to about 12 hours, about 6 hours to about 10hours; about 5 minutes to about 1 hour, about 5 minutes to about 30minutes; about 12 hours to about 1 week, about 24 hours to about 1 week,about 2 days to about 5 days, or about 3 days to about 5 days. In oneembodiment, the compound of the invention (e.g., a Factor XIa orkallikrein inhibitor) is administered as an intravenous infusion forabout 5, 10, 15, 30, 45, or 60 minutes or longer; about 1, 2, 4, 6, 8,10, 12, 16, or 24 hours or longer; about 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 days or longer.

Dosages and Dosing Regimens

The effective amount of a small molecule Factor XIa or kallikreininhibitor administered according to the present invention may bedetermined by one of ordinary skill in the art. The specific dose leveland frequency of dosage for any particular subject may vary and willdepend upon a variety of factors, including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the species, age, body weight, general health, sex and diet ofthe subject, the mode and time of administration, rate of excretion,drug combination, and severity of the particular condition.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination provided herewith may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level. Patients may, however,require intermittent treatment on a long-term basis upon any recurrenceof disease symptoms.

EXAMPLES

The compounds of the present invention are prepared as described in theCharts, Schemes, Preparation of Intermediates and Examples below, orprepared by methods analogous thereto, which are readily known andavailable to one of ordinary skill in the art of organic synthesis.

Synthesis of the Compounds of the Invention:

-   Temperatures are given in degrees Celsius (° C.). Unless otherwise    stated, operations are carried out at room or ambient temperature,    that is, at a temperature in the range of 18-25° C. under an inert    atmosphere with the exclusion of moisture. Chromatography means    flash chromatography on silica gel as described in Still, W. C,    Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923.; thin layer    chromatography (TLC) is carried out on silica gel plates. NMR data    is given in parts per million (ppm) relative to the deuterium lock    signal of the deuterated solvent utilized.-   Conventional abbreviations for signal shape are used. For mass    spectra (MS), the lowest mass major ion is reported for molecules    where isotope splitting results in multiple mass spectral peaks.    Solvent mixture compositions are given as volume percentages or    volume ratios. In cases where the NMR spectra are complex, only    diagnostic signals are reported.

Analytical HPLC

-   Method A: Agilent 1100 HPLC, Zorbax Eclipse XDB-C18 50×4.6 mm    column, 1.5 mL/min, Solvent A-Water (0.1% TFA), Solvent B    -Acetonitrile (0.07% TFA), Gradient -5 min 95%A to 90%B; 1 min.    hold; then recycle (to 95% A over 1 min), UV Detection @ 214 and 254    nm.-   Method B: Agilent 1100 HPLC, Zorbax Eclipse XDB-C18 50×4.6 mm    column, 1.5 mL/min, Solvent A-Water (0.1% TFA), Solvent B    -Acetonitrile (0.07% TFA), Gradient −5 min 95% A to 90% B; 1 min.    hold; then recycle (to 95% A over 1 min), UV Detection @ 210 and 254    nm.-   Method C: Agilent 1100 HPLC, Zorbax Eclipse XDB-C18 50×4.6 mm    column, 1.5 mL/min, Solvent A-Water (0.1% TFA), Solvent B    -Acetonitrile (0.07% TFA), Gradient −6 min 95% A to 95% B; 1 min.    hold; then recycle (to 95% A over 1 min), UV Detection @ 210 and 254    nm.    Terms and abbreviations:

ACN =acetonitrile,

BOC =Boc =tert-butoxycarbonyl,

br =broad,

t-BuOH =tert-butyl alcohol,

Cat. =catalytic,

Conc. =concentrated,

d =doublet,

dd =doublet of doublets,

ddd =doublet of doublet of doublets,

dt =doublet of triplets,

DCM =dichloromethane,

Dess-Martin periodinane=1,1,1-Tris(acetyloxy)1,1-dihydro-1,2-benziodoxol-3(1H)-one,

DIAD =diisopropyl azodicarboxylate,

DMF =N,N-dimethylforamide,

DMSO =dimethyl sulfoxide,

Et₂O =diethyl ether,

Et₃N =triethylamine,

EtOAc =ethyl acetate,

EtOH =ethyl alcohol,

equiv. =equivalent(s),

h =hour(s),

H₂O =water,

HCl =hydrochloric acid

HPLC =high performance liquid chromatography,

HOAc =acetic acid,

IPA =isopropyl alcohol,

ISCO =normal phase silica gel cartridges supplied by Teledyne ISCO,

K₂CO₃ =potassium carbonate,

LiBH₄ =lithium tetrahydroborate,

LiBr =lithium bromide,

LiCl =lithium chloride,

LAH =lithium tetrahydroaluminate,

m =multiplet,

min. =min =minute(s)

MgCl₂ =magnesium chloride

MeOH =methanol,

MsCl =methanesulfonyl chloride,

MTBE =methyl tert-butyl ether,

NaHCO₃ =sodium bicarbonate,

Na₂SO₄ =sodium sulfate,

NH₄OH =ammonium hydroxide,

NH₄OAc =ammonium acetate,

NH₄Cl =ammonium chloride,

NMR =nuclear magnetic resonance,

NMP =N-methylpyrrolidinone,

Pd-C =palladium on activated carbon

p =pentet,

PMB =p-methoxybenzyl,

PMBCl =p-methoxybenzyl chloride,

ret =retention

rt =room temperature,

s =singlet,

sat =saturated,

t =triplet,

TFA =trifluoroacetic acid,

TBDPS =t-butyldiphenylsilyl,

TBS =t-butyldimethylsilyl,

THF =tetrahydrofuran,

THP =tetrahydropyran,

TLC =thin layer chromatography

Example 1 General Synthetic Schemes and Methods

In general, in the instances when the compound of the invention containsa basic substituent (e.g., an amino group, pyridyl), an acidicsubstitutent (e.g., a carboxylic acid), or is zwitterionic (i.e.,containing both a basic substituent and an acidic substituent), for easeof isolation and handling, it is preferred to isolate the compound as asalt. This salt form facilitates characterization and is used directlyin biological assays.

-   Scheme 1 describes a general method for the preparation of (2S,    3R)-trans-disubstituted 4-oxoazetidine-2-carboxylates of general    structure A-8 and A-9. Alkylation of the dianion of N-protected    azetidinone A-1 (for example, R¹=TBS) with a commercially available    or readily prepared alkyl halide of general structure A-2 affords    the desired intermediate as predominately the trans product and with    retention of the stereochemistry at the 2-position (Baldwin, J. E.,    et al., Tetrahedron, 1990 46, 4733). For aminopyridine containing    compounds, the bromide described in the preparation of Intermediate    1 is preferred as alkylating agent. Further, the synthetic sequence    described for the preparation of Intermediate 1 is of general    utility for the preparation of a number of requisite bromides of    general structure A-2 from commercially available esters and    alcohols. Esterification of the carboxylic acid under standard    condensation conditions affords intermediate A-4 which is readily    purified by normal phase chromatography. For the temporary    protection of the carboxylic acid, the benzyl and 4-methoxybenzyl    esters are particularly useful. Removal of the N-protecting group of    the beta lactam affords the intermediate A-5 which is readily    acylated with a commercially available or readily prepared    isocyanate A-6 (for example, Tsai, M. H., Takaoka, L. R., Powell, N.    A., Nowick, J. S. Org. Syn. 2002, 78, 220) to afford the beta lactam    intermediate A-8. Intermediate A-5 can also be converted to A-8 by    its reaction with a number of other reagents known to react with    amines to afford ureas such as phenyl carbamates (A-7 where    Ar=phenyl, for example: Thavonekham, B. Synthesis, 1997, 1189) and    4-nitrophenyl carbamates (A-7 where Ar is 4-nitrophenyl, for    example: Hutchins, S. M., Chapman, K. T. Tet. Lett. 1994, 35, 4055).    Additionally, reaction of A-5 with a carbamoyl chloride (A-10 where    R⁵=Me, for example: Holmes, D. L., Smith, E. M., Nowick, J. S. J.    Am. Chem. Soc. 1997, 119(33), 7665) affords tetra-substituted ureas    A-8 (R⁵=Me). Commercially available or readily prepared amines,    R⁴-NH₂, (for a recent review on the asymmetric synthesis of amines    of particular interest see, Robak, M. T., Herbage, M. A.,    Ellman, J. A. Chem. Rev. 2010, 110, 3600) serve as suitable starting    materials for the preparation of isocyanates, A-6 and carbamates,    A-7. Removal of the temporary protecting group of the carboxylate    (either by hydrogenation in the cases where R³ is benzyl or via    acidolysis with TFA when R³ is 4-methoxybenzyl) and removal of any    other protecting groups present in the molecule affords the examples    of general structure A-9. In those examples where the ester CO₂R³ is    retained, removal of any other protecting groups present in the    molecules affords examples of general structure A-8. For some    structures A-9, the carboxylic acid can also be esterified using    methods known to one skilled in the art to afford additional    examples of general structure A-8. Exemplary compounds of the    present invention synthesized according to the route described in    Scheme 1 are summarized in Chart A (FIGS. 1A-1M).

-   Scheme 2 describes a general method for the preparation of    2,3-disubstituted 4-oxoazetidines containing a 2-cyano group.    Conversion of the carboxylic acid in intermediate B-1 (prepared as    described for intermediate A-3 in Scheme 1) to the carboxamide (for    example by the action of di-tert-butyldicarbonate and ammonium    bicarbonate in the presence of pyridine as described by Pozdnev, V.,    Tet. Lett., 1995, 36, 7115) affords intermediate B-2 with    concomitant removal of the silyl protecting group at N-1.    Dehydration of the aliphatic amide provides the nitrile intermediate    B-3 (for a general review, see Mowry, D., Chem. Rev. 1948, 42, 189).    Reaction with an isocyanate B-4 and, if necessary, removal of any    protecting groups remaining affords examples of the general formula    B-5. Exemplary compounds of the present invention synthesized    according to the route described in Scheme 2 are summarized in Chart    B (FIG. 2).

-   Scheme 3 describes the synthesis of    3-[2-(2-amino-1,3-thiazol-5-yl)ethyl]azetidine-2-ones of general    structure C-8. Oxidation of alkene C-2 (readily prepared by the    methods described in Scheme 1) affords aldehyde C-3 which is treated    successively with tetrabutylammonium bromide followed by thiourea to    afford the aminothiazole intermediate C-4 with concomitant removal    of the protecting group at N-1. Protection of the aminothiazole with    a suitable protecting group such as BOC affords intermediate C-5.    Reaction with an isocyanate C-6 followed by removal of any    protecting groups affords examples of the general formula C-8.    Exemplary compounds of the present invention synthesized according    to the route described in Scheme 3 are summarized in Chart C and D    (FIG. 3).

-   Scheme 4 describes the synthesis of    3-(2-amino-thiazol-5-ylmethyl)azetidine-2-ones of general structure    D-8. Treatment of vinyl halide D-2 (readily prepared by the methods    described in Scheme 1) with MCPBA followed by condensation of the    intermediate chloro epoxide with thiourea affords the aminothiazole    intermediate D-3. Protection of the aminothiazole with a suitable    group such as BOC affords intermediate D-4. Deprotection of the beta    lactam nitrogen and reaction with an isocyanate D-6 affords    intermediate D-7. Removal of any remaining protecting groups affords    examples of the general formula D-8. Exemplary compounds of the    present invention synthesized according to the route described in    Scheme 4 are summarized in Charts C and D (FIG. 3).

-   Scheme 5 describes a general method for the preparation of    2-carboxamides of general structure E-6. Condensation of acid E-1    (prepared as described for A-3 of Scheme 1) affords amide E-2.    Removal of the N-1 protecting group followed by condensation of    isocyanate E-4 affords intermediate E-5. Removal of the amide    protecting group and any other protecting groups present in the    molecule affords examples of general formula E-6. Exemplary    compounds of the present invention synthesized according to the    route described in Scheme 5 are summarized in Chart E (FIG. 4).

-   Scheme 6 describes the synthesis of (2R,3R)-trans-disubstituted    ethers of the general formula F-8. Oxidative decarboxylation of F-1    (prepared as described for A-3 of Scheme 1) affords the benzoate F-2    (for example, see Shiozaki, M., Synthesis, 1990, 691). Removal of    the N-1 protecting group followed by displacement of the benzoate    with an oxygen nucleophile, F-5, affords ether F-6. Reaction with an    isocyanate F-7 and removal of any protecting groups present in the    molecule affords examples of general formula F-8. Exemplary    compounds of the present invention synthesized according to the    route described in Scheme 6 are summarized in Chart F (FIG. 5).

-   Scheme 7 describes the synthesis of sulfones of general formula G-4.    Reaction of benzoate G-1 (prepared as described for F-3, Scheme 6)    with sodium methanesulfinate affords the sulfone G-2 (Clauss, K.,    et. al. Liebigs Ann. Chem. 1974, 539). Reaction with an isocyanate    G-3 and subsequent removal of any protecting groups present in the    molecule affords examples of general formula G-4. Exemplary    compounds of the present invention synthesized according to the    route described in Scheme 7 are summarized in Chart G (FIG. 6).

-   Scheme 8 describes a general method for the preparation of oximes of    general structure H-9. Reduction of N-protected azetidinone H-1 (for    example, R¹=TBDPS) affords the hydroxymethyl intermediate H-2 which    is readily oxidized to the aldehyde H-3. Condensation with    methoxyamine affords the oxime H-4 as a mixture of geometric    isomers. Alkylation of the anion of oxime H-4 with a commercially    available or readily prepared alkyl halide of general structure H-5    affords the 2,3-trans product H-6. Removal of the N-protecting group    of the beta lactam affords the intermediate H-7. Reaction with an    isocyanate H-8 and removal of any protecting groups present in the    molecule affords examples of general formula H-9. Exemplary    compounds of the present invention synthesized according to the    route described in Scheme 8 are summarized in Chart H (FIG. 7).

-   Scheme 9 describes a general method for the preparation of examples    of general structure I-4 through I-7. Removal of the Boc and PMB    protecting groups of I-1 (prepared as described for A-8, Scheme 1)    affords the aminopyridine I-2. Reaction with a chloroformate or    other suitable carbonic acid derivative affords carbamate of general    structure I-4. Removal of the carboxylic acid group, if desired,    affords compounds of general structure I-5. Further, reaction of I-2    with a carboxylic acid R⁷CO₂H in the presence of a suitable    condensation agent affords compounds of general structure I-6.    Removal of the carboxylic acid protecting group and condensing with    a different alcohol R³OH in the presence of a suitable condensing    agent allows for the interconversion of the R³ group of I-6. In    those cases where the free carboxylic acid is desired, removal of    the carboxylic acid protecting group and any other protecting groups    present in the molecule affords compounds of general structure I-7.    Exemplary compounds of the present invention synthesized according    to the route described in Scheme 9 are summarized in Chart I (FIG.    8).

-   Scheme 10 describes a general method for the preparation of examples    of general structure J-3 and J-4. Transient silylation of compounds    of general structure J-1 (prepared as described in Scheme 1)    followed by reaction with nitrophenyl carbonate J-2 (R⁵=H or Me)    affords the compound of general structure J-3. Reaction of J-3 with    an alcohol (R³-OH) in the presence of a suitable condensing agent or    a bromide (R³-Br) in the presence of a suitable base provides esters    of general structure J-4. Exemplary compounds of the present    invention synthesized according to the route described in Scheme 10    are summarized in Chart J (FIGS. 9A-9B).

-   Scheme 11 describes a general method for the preparation of (2S,    3R)-trisubstituted 4-oxoazetidine-2-carboxylates of general    structure K-9. The chemistry is completely analogous to the method    described in Scheme 1 in which the starting material utilized is the    known lactam K-1 (see Finke, P. E., et. al., J. Med. Chem. 1995, 38,    2449). Exemplary compounds of the present invention synthesized    according to the route described in Scheme 11 are summarized in    Chart K (FIGS. 10A-10B).

Example 2 Preparation of Intermediates Intermediate 1: tert-Butyl[4-(bromomethyl)pyridin-2-yl](4-methoxybenzyl)carbamate

Step 1. Preparation of Methyl2-[(tert-butoxycarbonyl)amino]isonicotinate

A solution of di-tert-butyldicarbonate (47.8 g, 219 mmol) in t-BuOH (650mL, 6.80 mol) was warmed at 33° C. and treated with methyl2-aminoisonicotinate (30.4 g, 200 mmol) in portions over 1 h [˜5 g addedevery 10 min]. The reaction mixture was stirred at 33° C. overnight (18h); HPLC/LC MS indicated nearly complete conversion. The solids werecollected by vacuum filtration [course frit sintered glass funnel] andwashed with Et₂O (˜200 mL). The light tan solid was dried on high vacfor 3.5 h until constant weight was achieved (39.65 g, 79%): MS (ESI+)for C₁₂H₁₆N₂O₄ m/z 253.2 [M+H]⁺, 275.2 [M+Na]⁺; HPLC retention time:3.25 min (Method B).

Step 2. Preparation of tert-butyl[4-(hydroxymethyl)pyridin-2-yl](4-methoxybenzyl)carbamate

A slurry of methyl 2-[(tert-butoxycarbonyl)amino]isonicotinate (41.5 g,164 mmol) in DMF (610 mL, 7.90 mol) was cooled to ˜0° C. (ice/NaCl) andtreated with a 1.0 M solution of NaHMDS in THF (197 mL, 197 mmol)dropwise over 2 h to afford a clear, brown solution. The reactionmixture was stirred for 30 min at 0° C. and treated with PMBCl (24.5 mL,181 mmol) dropwise over 11 min. The reaction mixture was stirred for 10min to afford a yellow/brown mixture, the cold bath was removed, and thereaction mixture stirred at rt for 1 h 40 min; HPLC indicated nearlycomplete conversion. The reaction mixture was cooled to ˜0° C.(ice/NaCl) and the reaction was quenched by the addition of saturatedaqueous NH₄Cl (100 mL). The mixture was allowed to warm to rt and wasextracted with Et₂O (1×200 mL). The separated aqueous layer was dilutedwith water (100 mL) and extracted with Et₂O (2×100 mL). The combinedorganics were washed with saturated aqueous NaHCO₃ (1×400 mL) and theseparated aqueous layer was filtered [course sintered glass] andextracted with Et₂O (2×100 mL). The combined organics were washed with10% aqueous LiCl (2×600 mL), dried (Na₂SO₄), and concentrated in vacuoto afford an orange oil. The crude material was further dried on highvac overnight to afford a light colored solid/orange oil (80.86 g),which was carried on without further manipulation: MS (ESI+) forC₂₀H₂₄N₂O₅ m/z 373.2 [M+H]⁺; HPLC retention time: 4.53 min (Method B);¹H NMR indicated the material was contaminated withhexamethyldisilazane. The above crude methyl2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]isonicotinate (80.86 g)was divided into two equal portions and each was treated as follows: Asolution of crude material in THF (400 mL) was cooled to ˜0° C.(ice/NaCl) and treated with LiBH₄ (3.94 g, 181 mmol) in one portion. Thereaction mixture was stirred for 5 min, the cold bath was removed, andthe reaction mixture was allowed to warm to rt with stirring over 30min. The flask was transferred to a preheated 50° C. oil bath and thereaction mixture was stirred at 40° C. for 2.5 h; HPLC indicatedcomplete conversion. The reaction mixture was cooled to ˜0° C. and thereaction was carefully quenched by the slow, dropwise addition ofsaturated aqueous NaHCO₃ (100 mL). The mixture was diluted with water(150 mL) and extracted with EtOAc (1×200 mL, 2×100 mL). The combinedorganics were washed with saturated aqueous NH₄Cl (1×200 mL), water (1×200 mL), and brine (1×200 mL), dried (Na₂SO₄), and concentrated invacuo to afford the title compound as a viscous yellow oil (55.6 g,89%), which was carried on without further manipulation: MS (ESI+) forC₁₉H₂₄N₂O₄ m/z 345.3 [M+H]⁺; HPLC retention time: 3.19 min (Method B).

Step 3. Preparation of tert-Butyl[4-(bromomethyl)pyridin-2-yl](4-methoxybenzyl)carbamate

To a stirring solution of tert-butyl[4-(hydroxymethyl)pyridin-2-yl](4-methoxybenzyl)carbamate (23.14 g,67.19 mmol) in THF (300 mL) cooled in an ice/water bath was added Et₃N(13.6 mL, 97.8 mmol). MsCl (7.20 mL, 93.1 mmol) was added slowly over 15min and the reaction mixture was stirred for 1 h at 0° C.; HPLCindicated nearly complete conversion. LiBr (8.54 g, 98.3 mmol) was addedin one portion and the cold bath was removed. The reaction mixture wasstirred overnight at rt; HPLC indicated complete conversion to thedesired product. The reaction mixture was partitioned between EtOAc (250mL) and water (250 mL). The separated aqueous phase was extracted withEtOAc (200 mL) and the combined organic phases were washed with water(300 mL), dried (MgSO₄), and evaporated in vacuo to afford a viscousorange oil (12.05 g). The crude mixture was adsorbed onto silica andpurified by flash column chromatography (500 g silica, packed withhexanes, eluted with 15% EtOAc/hexanes) to afford the title compound asa pale yellow oil (11.59 g, 42%): MS (ESI+) for C₁₉H₂₃BrN₂O₃ m/z 407.1,409.1 [M+H]⁺; HPLC ret. time: 4.48 min (Method B); ¹H NMR (CDCl₃, 300MHz) δ8.42-8.30 (m, 1H), 7.73 (br s, 1H), 7.26-7.19 (m, 2H), 7.07-6.9.9(m, 1H), 6.85-6.77 (m, 2H), 5.14 (s, 2H), 4.38 (s, 2H), 3.78 (s, 3H),1.44 (s, 9H).

Intermediate 2: 1-[(1R)-1-isocyanatoethyl]-3-(trifluoromethoxy)benzene

To a cooled (0-5° C.), vigorously stirred solution of(1R)-1-[3-(trifluoromethoxy)phenyl]ethanamine hydrochloride (prepared asdescribed in steps 1 through 3 in Example 4 from3-trifluoromethoxy-benzaldehyde) (0.197 g, 0.815 mmol) in methylenechloride (4 mL) and sat NaHCO₃ (4 ml) was added triphosgene (0.080 g,0.269 mmol) in one portion. After 30 min, the mixture was transferred toa separatory funnel and the organic layer collected. The aqueous layerwas washed with DCM and the combined organic layers were dried withanhydrous sodium sulfate, filtered and concentrated to afford the titlecompound (155 mg, 82%) as a viscous oil which was used immediatelywithout further purification. ¹H NMR (300 MHz, CDCl₃) δ1.63 (d, J=7 Hz,3H), 4.82 (q, J=7 Hz, 1H), 7.16-7.29 (m, 3H), 7.41 (t, J=8 Hz, 1H).

Intermediate 3. 4-methoxybenzyl(2S,3R)-3-({2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-4-oxo-1-{[(1S)-2,2,2-trifluoro-1-phenylethyl]carbamoyl}azetidine-2-carboxylate

To a solution of 4-nitrophenylchloroformate (0.378 g, 2.22 mmol) intetrahydrofuran (20 mL) at ambient temperature was added a solution of(S)-2,2,2-trifluoro-1-phenyl-ethylamine (0.390 g, 2.22 mmol) andpyridine (0.180 mL, 2.22 mmol) in THF (20 mL) dropwise over 45 min.After 15 min, the mixture was diluted with ethyl acetate and washed with0.1 N aqueous HCl, brine, dried with anhydrous sodium sulfate, filtered,and concentrated to afford 4-nitrophenyl[(1S)-2,2,2-trifluoro-1-phenylethyl]carbamate which was used withoutfurther purification. MS (ESI+) for C₁₅H₁₁F₃N₂O₄ m/z 341.3 (M+H)⁺; HPLCretention time: 4.4 min (Method C). To a solution of the carbamatedescribed above in THF (5 mL) was added 4-methoxybenzyl(2S,3R)-3-({2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-4-oxoazetidine-2-carboxylate(185 mg, 0.329 mmol), triethylamine (0.080 mL, 0.57 mmol) and4-dimethylaminopyridine (0.002 g, 0.016 mmol) at ambient temperature.After 3 days, volatiles were removed at reduced pressure and the residuepurified by flash chromatography using hexanes and ethyl acetate(15-30%) as eluent to afford the title compound (220 mg, 74%) as a whitesolid which contained approximately 15% of(S,S)-1,3-bis-(2,2,2-trifluoro-1-phenyl-ethyl)-urea that was readilyremoved by chromatography after removal of the protecting groups. ¹H NMR(300 MHz, CDCl₃) δ1.43 (s, 9H), 3.02-3.24 (m, 2H), 3.50-3.56 (m, 1H),3.78 (s, 3H), 3.80 (s, 3H), 4.29 (d, J=3 Hz, 1H), 5.50-5.62 (m, 1H),6.79-6.85 (overlapping m, 5H), 7.10 (d, J=9 Hz, 2H), 7.21-7.23(overlapping m, 3H), 7.39-7.45 (overlapping m, 6H), 7.64 (s, 1H), 8.27(d, J=5 Hz, 1H); MS (ESI+) for C₄₀H₄₁F₃N₄O₈ m/z 763.0 (M+H)⁺; HPLCretention time: 5.54 min (Method C).

Intermediate 4. tert-Butyl[4-(2-bromoethyl)pyridin-2-yl](4-methoxybenzyl)carbamate

Step 1. Preparation of tert-butyl (4-methylpyridin-2-yl)carbamate

A solution of 2-pyridinamine, 4-methyl-(0.60 g, 5.5 mmol) in t-BuOH (35mL) was treated with di-tert-butyldicarbonate (1.3 g, 6.1 mmol). Thereaction mixture was stirred at 30° C. for 2 d 17 h; HPLC/TLC indicatedconversion to product. The solvent was removed in vacuo to afford a tancrystalline solid, which was recrystallized from hot isopropanol to givethe title compound as a white crystalline solid (0.87 g, 75%): (¹H NMRconsistent, does not show any contamination, which agrees with the LC MSthough HPLC shows two peaks) MS (ESI+) for C₁₁H₁₆N₂O₂ m/z 153.2[M-tBu+H]⁺; HPLC retention time: 2.24 min (second peak at 2.11 min)(Method B).

Step 2. Preparation of tert-butyl(4-methoxybenzyl)(4-methylpyridin-2-yl)carbamate

A solution of tert-butyl (4-methylpyridin-2-yl)carbamate (81 mg, 0.39mmol) in DMF (1.2 mL) was cooled to −10° C. (ice/brine) and treated withsodium hydride (60% in mineral oil, 22 mg, 0.55 mmol). The reactionmixture was vigorously stirred for 20 min at −10° C. to afford a nearlyhomogeneous solution, which was treated with PMBCl (0.07 mL, 0.5 mmol).The reaction mixture was stirred for 40 min to afford a pink mixture,the cold bath was removed, and the mixture was stirred at rt for 17 h toafford an orange mixture; HPLC/LC MS indicated complete conversion toproduct. The reaction was quenched by the addition of water (1 mL) anddiluted with water (5 mL) and Et₂O (40 mL). The separated organic layerwas washed with water (10 mL), 0.1 N aqueous HCl (10 mL), saturatedaqueous NaHCO₃ (10 mL), and brine (10 mL), dried (Na₂SO₄), andconcentrated in vacuo to afford a light orange oil. Purification bycolumn chromatography (2×8 cm silica; Hex, 5%, 10%, 20% EtOAc/Hex)afforded the title compound as a clear, colorless oil (91 mg, 71%); MS(ESI+) for C₁₉H₂₄N₂O₃ m/z 329.2 [M+H]⁺; HPLC retention time: 3.66 min(Method B).

Step 3. Preparation of ethyl{2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}acetate andtert-butyl 2-[(4-methoxybenzyl)amino]-4-methylnicotinate

A solution of tert-butyl(4-methoxybenzyl)(4-methylpyridin-2-yl)carbamate (3.3 g, 10.0 mmol) anddiethyl carbonate (6.1 mL, 50.0 mmol) in THF (82 mL) was cooled to −78°C. (acetone/CO₂) and treated dropwise with a 1.33 M solution of LDA inhexanes/THF/ethylbenzene (9.1 mL, 12 mmol) over 15 min. Theyellow-orange reaction mixture was stirred for 30 min at −78° C.; HPLCindicated a 1.1:1:0.5 mixture of tert-butyl(4-methoxybenzyl)(4-methylpyridin-2-yl)carbamate, desired product ethyl{2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl} acetateand side product tert-butyl2-[(4-methoxybenzyl)amino]-4-methylnicotinate. At 45 min the reactionwas quenched by the addition of acetic acid (0.68 mL, 12 mmol) and themixture was diluted with water (75 mL) and EtOAc (36 mL). The mixturewas allowed to warm to rt and the separated aqueous layer was extractedwith EtOAc (2×36 mL). The combined organics were dried (Na₂SO₄) andconcentrated in vacuo to afford a yellow oil. Purification by columnchromatography (5×18 cm silica; Hex, 5%, 10%, 15%, 20% EtOAc/Hex)afforded a 70:30 mixture of ethyl{2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl} acetateand tert-butyl 2-[(4-methoxybenzyl)amino]-4-methylnicotinate as a clear,colorless oil (1.77 g, 33%) and recovered tert-butyl(4-methoxybenzyl)(4-methylpyridin-2-yl)carbamate as a clear, colorlessoil (1.78 g, 54%): ¹H NMR indicated a 2.9:1 mixture of ethyl{2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}acetate andtert-butyl 2-[(4-methoxybenzyl)amino]-4-methylnicotinate; MS (ESI+) forC₂₂H₂₈N₂O₅ m/z 401.3 [M+H]⁺, MS (ESI+) for C₁₉H₂₄N₂O₃ m/z 329.3 [M+H]+;HPLC ret. time for ethyl{2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}acetate:4.22 min (Method B), HPLC retention time for tert-butyl2-[(4-methoxybenzyl)amino]-4-methylnicotinate: 3.24 min (Method B).

Step 4. Preparation of tert-butyl[4-(2-hydroxyethyl)pyridin-2-yl](4-methoxybenzyl)carbamate

A ˜2:1 mixture of ethyl{2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}acetate(1.77 g, 3.27 mmol) and tert-butyl2-[(4-methoxybenzyl)amino]-4-methylnicotinate in THF (16 mL) was cooledto 0° C. (ice/brine) and treated with LiBH₄ (0.21 g, 9.6 mmol). The coldbath was removed and the reaction mixture was allowed to warm to rt over20 min, followed by heating at 50° C. for 3 h; HPLC/LC MS indicatedcomplete consumption of{2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}acetate.The reaction mixture was allowed to cool to rt and the reaction wascarefully quenched by the addition of saturated aqueous NaHCO₃ (30 mL).The mixture was diluted with water (60 mL) and extracted with EtOAc(3×60 mL). The combined organics were dried (Na₂SO₄) and concentrated invacuo to afford a bright yellow oil. Purification by columnchromatography (4×14 cm silica; Hex, 10%, 30%, 50% EtOAc/Hex) affordedthe title compound as a clear, colorless oil (0.91 g, 78%): MS (ESI+)for C₂₀H₂₆N₂O₄ m/z 359.3 [M+H]+; HPLC retention. time: 3.17 min (MethodB).

Step 5. Preparation of tert-Butyl[4-(2-bromoethyl)pyridin-2-yl](4-methoxybenzyl)carbamate

A mixture of tert-butyl[4-(2-hydroxyethyl)pyridin-2-yl](4-methoxybenzyl)carbamate (0.91 g, 2.5mmol) and carbon tetrabromide (0.93 g, 2.8 mmol) in CH₂Cl₂ (15 mL) wascooled to 0° C. (ice/water). Triphenylphosphine (0.73 g, 2.8 mmol) wasadded in one portion and the bright yellow reaction mixture was stirredfor 30 min at 0° C. The cold bath was removed and reaction mixture wasstirred at rt for 1 h; HPLC indicated nearly complete conversion toproduct. At 2 h, HPLC indicated no change. The reaction mixture wasdiluted with CH₂Cl₂ (120 mL) and washed with saturated aqueous NaHCO₃(70 mL). The separated aqueous phase was extracted with CH₂Cl₂ (2×70 mL)and the combined organics were dried (Na₂SO₄) and concentrated in vacuoto afford a clear, colorless oil. Purification by column chromatography(4×16 cm silica; Hex, 10%, 20%, 30% EtOAc/Hex) afforded the titlecompound as a clear, colorless oil (0.69 g, 64%): MS (ESI+) forC₂₀H₂₅BrN₂O₃ m/z 421.2 [M+H]⁺; HPLC retention. time: 4.29 min (MethodB); ¹H NMR (CDCl₃, 300 MHz) δ8.37-8.28 (m, 1H), 7.54 (br s, 1H),7.26-7.16 (m, 2H), 6.90-6.84 (m, 1H), 6.84-6.74 (m, 2H), 5.38-4.88 (m,2H), 3.77 (s, 3H), 3.62-3.51 (m, 2H), 3.19-3.09 (m, 2H), 1.44 (s, 9H).

Intermediate 5: tert-Butyl [4-(bromomethyl)pyridin-2-yl]methylcarbamate

Step 1. Preparation of methyl2-[(tert-butoxycarbonyl)(methyl)amino]isonicotinate

A slurry of methyl 2-[(tert-butoxycarbonyl)amino]isonicotinate (5.0 g,2.0E1 mmol) in DMF (75 mL) was cooled to ˜0° C. (ice/NaCl) and treatedwith a 1.0 M solution of sodium hexamethyldisilazane in THF (24 mL, 24mmol) dropwise over 25 min to afford a clear, yellow/brown solution. Thereaction mixture was stirred for 30 min at 0° C. and treated with methyliodide (1.4 mL, 22 mmol). The reaction mixture was stirred for 20 min,the cold bath was removed, and the reaction mixture stirred at rt for 2h; HPLC/LC MS indicated complete conversion to product. The reactionmixture was cooled to ˜0° C. and the reaction was quenched by theaddition of saturated aqueous NH₄Cl (25 mL). The mixture was allowed towarm to rt and was extracted with Et₂O (3×50 mL). The combined organicswere washed with saturated aqueous NaHCO₃ (1×50 mL) and 10% aqueous LiCl(2×50 mL), dried (Na₂SO₄), and concentrated in vacuo to afford a brightyellow oil with precipitate. The crude material was further dried onhigh vacuum overnight to afford the crude title compound as a lightcolored solid/yellow oil (5.29 g). MS (ESI+) for C₁₃H₁₈N₂O₄LC m/z 267.1(M+H)⁺; HPLC retention time: 3.78 min (Method B).

Step 2. Preparation of tert-Butyl[4-(hydroxymethyl)pyridin-2-yl]methylcarbamate

A solution of crude methyl2-[(tert-butoxycarbonyl)(methyl)amino]isonicotinate (5.29 g) in THF (50mL) was cooled to ˜0° C. (ice/brine) and treated with lithiumtetrahydroborate (0.47 g, 22 mmol) in one portion. The reaction mixturewas stirred for 10 min, the cold bath was removed, and the reactionmixture was allowed to warm to rt over 30 min. The flask was transferredto a preheated 40° C. oil bath and the reaction mixture was stirred at40° C. for 3.5 h; HPLC indicated a mixture of product and startingmaterial. The oil bath temperature was increased to 50° C. for 50 min;HPLC indicated nearly complete conversion to product. The reactionmixture was allowed to cool to rt and was stored at 0-5° C. overnight.The reaction mixture was cooled to ˜0° C. and the reaction was carefullyquenched by the slow, dropwise addition of saturated aqueous NaHCO₃ (15mL). The mixture was diluted with water (20 mL) and extracted with EtOAc(3×20 mL). The combined organics were washed with saturated aqueousNH₄Cl (1×20 mL), water (1×20 mL), and brine (1×30 mL), dried (Na₂SO₄),and concentrated in vacuo to afford the crude title compound as a tanoil (4.49 g). MS (ESI+) for C₁₂H₁₈ N₂O₃LC m/z 239.2 (M+H)⁺; HPLCretention time: 2.10 min (Method B).

Step 3. Preparation of tert-Butyl[4-(bromomethyl)pyridin-2-yl]methylcarbamate

A mixture of crude tert-butyl[4-(hydroxymethyl)pyridin-2-yl]methylcarbamate (4.49 g) and carbontetrabromide (7.2 g, 22 mmol) was taken up in CH₂Cl₂ (110 mL) and cooledto 0° C. (ice water). Triphenylphosphine (5.7 g, 22 mmol) was added inone portion and the bright yellow reaction mixture was stirred for 20min, the cold bath was removed, and the orange-red mixture was stirredat rt for 1 h; HPLC/LC MS indicated complete conversion to product. Thereaction mixture was diluted with CH₂Cl₂ (50 mL) and washed withsaturated aqueous NaHCO₃ (1×50 mL). The separated aqueous layer wasextracted with CH₂Cl₂ (2×20 mL) and the combined organics were dried(Na₂SO₄) and concentrated in vacuo to afford a red-orange oil, which wasstored at 0-5° C. overnight. Purification by column chromatography (5×14cm silica; Hex, 10%, 20%, 30% EtOAc/Hex) afforded the title compound asa clear, nearly colorless oil (4.74 g, 80%). MS (ESI+) for C₁₂H₁₇BrN₂O₂m/z 301.1, 303.1 (M+H)⁺; HPLC retention time: 3.52 min (Method B); ¹HNMR (300 MHz, CDCl₃) δ8.43-8.27 (m, 1H), 7.84-7.66 (m, 1H), 7.12-6.92(m, 1H), 4.39 (br s, 2H), 3.41 (s, 3H), 1.54 (s, 9H).

Example 3 Scheme 1:(2S,3R)-3-[(2-Aminopyridin-4-yl)methyl]-1-{[(1R)-1-cyclohexylethyl]carbamoyl}-4-oxoazetidine-2-carboxylicacid trifluoroacetate

Step 1. Preparation of(2S,3R)-3-({2-[tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylicacid

An oven-dried, 3-neck flask was cooled under nitrogen, charged with(2S)-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylic acid(3.00 g, 13.1 mmol) and dry THF (110 mL), and the mixture was cooled to−70° C. Lithium diisopropylamide in THF [1.25 M (titrated), 21.4 mL,26.8 mmol] was added dropwise over 8 min., keeping the internaltemperature below −55° C., and the resulting tan mixture was stirred at−78° C. for 35 min and then placed in an ice-brine-MeOH bath and stirredbelow −15° C. for an additional 30 min. A solution of tert-butyl[4-(bromomethyl)pyridin-2-yl](4-methoxybenzyl)carbamate (5.86 g, 14.4mmol) in dry THF (21 mL) which had been cooled to 0° C. was then addedover 5 min., keeping the internal temperature below −5° C., and theresulting dark mixture was stirred at this temperature for 1 h, quenchedwith aqueous citric acid (0.5 M, 120 mL), diluted with water (120 mL)and extracted with EtOAc (3×100 mL). The combined organic phase waswashed with brine (60 mL), dried over anhydrous magnesium sulfate,filtered and concentrated under reduced pressure to give the crudeproduct as a clear, tacky residue which was used as is in the next step.MS (ESI+) for C₂₉H₄₁N₃O₆Si m/z 556.5 (M+H)⁺.

Step 2. Preparation of benzyl(2S,3R)-3-({2-[tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylate

A stirred solution of crude(2S,3R)-3-({2-[tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylicacid in DCM (60 mL) under nitrogen was treated withN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (3.26 g,17.0 mmol), followed by benzyl alcohol (1.62 mL, 15.7 mmol) and4-dimethylaminopyridine (160 mg, 1.31 mmol), and the resulting brownmixture was stirred at rt for 2 h, at which point HPLC indicated thereaction was complete. The mixture was diluted with water (60 mL) andextracted with DCM (2×60 mL), and the combined organic phase was washedwith water (60 mL) and brine (50 mL), dried over Na₂SO₄, filtered andconcentrated under reduced pressure. Purification by silica gelchromatography [120 g cartridge; elution with a gradient of 10-20%EtOAc/hexanes] provided 4.16 g of the title compound (contaminated with˜15wt % benzyl alcohol; 42% yield over first two steps) as a tan,viscous oil which was used without further purification. MS (ESI+) forC₃₆H₄₇N₃O₆Si m/z 646.7 (M+H)⁺.

Step 3. Preparation of benzyl(2S,3R)-3-({2-[tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-4-oxoazetidine-2-carboxylate

A stirred solution of benzyl(2S,3R)-3-({2-[tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylate(2.98 g, 3.92 mmol) in MeOH (39 mL) under nitrogen was treated withacetic acid (0.780 mL, 13.7 mmol) followed by a solution of ammoniumfluoride in MeOH (0.5 M, 10.2 mL, 5.10 mmol), and the resultinghomogeneous mixture was stirred at rt for 1 h, at which point HPLCindicated the reaction was complete. The mixture was concentrated underreduced pressure, and the residue was taken up in DCM (120 mL), washedwith sat aqueous NaHCO₃ (80 mL), water (60 mL) and brine (40 mL), driedover Na₂SO₄, filtered and concentrated under reduced pressure.Purification by silica gel chromatography [40 g cartridge; elution with20-50% EtOAc/hexanes] afforded 1.95 g (94%) of the title compound as awhite solid. MS (ESI+) for C₃₀H₃₃N₃O₆ m/z 532.4 (M+H)⁺.

Step 4. Preparation of benzyl(2S,3R)-3-({2-[tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-1-{[(1R)-1-cyclohexylethyl]carbamoyl}-4-oxoazetidine-2-carboxylate

A stirred solution of benzyl(2S,3R)-3-({2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-4-oxoazetidine-2-carboxylate(2.72 g, 5.12 mmol) in DCM (51 mL) under nitrogen was treated with Et₃N(2.50 mL, 17.9 mmol) followed by a solution of commercial[(1R)-1-isocyanatoethyl]cyclohexane (1.02 g, 6.65 mmol) in DCM (5 mL),and the mixture was stirred at rt overnight, at which point HPLCindicated starting material was nearly consumed. At 17 h, the mixturewas concentrated under reduced pressure, and the residue was purified bysilica gel chromatography [120 g; elution with 15-25% EtOAc/hexanes] togive 2.31 g (66%) of the title compound as a white foam. MS (ESI+) forC₃₉H₄₈N₄O₇ m/z 685.7 (M+H)⁺.

Step 5. Preparation of(2S,3R)-3-({2-[tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-1-{[(1R)-1-cyclohexylethyl]carbamoyl}-4-oxoazetidine-2-carboxylicacid

A mixture of benzyl(2S,3R)-3-({2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-1-{[(1R)-1-cyclohexylethyl]carbamoyl}-4-oxoazetidine-2-carboxylate(1.94 g, 2.83 mmol) in 1:1 MeOH/EtOAc (56 mL) under nitrogen was treatedwith 10% palladium-on-carbon (301 mg, 0.283 mmol Pd), and the mixturewas evacuated and filled with nitrogen twice and then stirred under ahydrogen atmosphere (double-layer balloon) for 70 min, at which pointHPLC indicated the reaction was complete. The catalyst was filtered offthrough solka floc, rinsing with 1:1 MeOH/EtOAc, and the filtrate wasconcentrated under reduced pressure to give 1.67 g (99%) of the titlecompound as a white, tacky solid. MS (ESI+) for C₃₂H₄₂N₄O₇ m/z 595.5(M+H)⁺.

Step 6. Preparation of(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-1-{[(1R)-1-cyclohexylethyl]carbamoyl}-4-oxoazetidine-2-carboxylicacid trifluoroacetate

A stirred solution of(2S,3R)-3-({2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-1-{[(1R)-1-cyclohexylethyl]carbamoyl}-4-oxoazetidine-2-carboxylicacid (78.0 mg, 0.131 mmol) in DCM (2.1 mL) under nitrogen was cooled inan ice-water bath and treated dropwise with TFA (0.7 mL, 9 mmol). Theresulting mixture was stirred at 0-5° C. for 30 min and then warmed tort and stirred overnight, at which point HPLC indicated the reaction wascomplete. The mixture was concentrated under reduced pressure, and theresidue was purified by preparative HPLC on a CombiFlash Rf system [30 gC18 Gold column; elution with a gradient of 10% acetonitrile (0.07%TFA)/water (0.1% TFA) to 100% acetonitrile (0.07% TFA)]. Lyophilizationof product fractions afforded 45 mg (70%) of the title compound as awhite amorphous solid. ¹H NMR (400 MHz, MeOD) δ7.79 (d, J=6.4 Hz, 1H),6.98 (s, 1H), 6.91 (d, J=6.8 Hz, 1H), 6.64 (bd, J=9.2 Hz, 1H), 4.27 (d,J=2.8 Hz, 1H), 3.70 (m, 2H), 3.24 (m, 2H), 1.85-1.65 (m, 5H), 1.42 (m,1H), 1.36-1.15 (m, 3H), 1.17 (d, J =6.8 Hz, 3H), 1.15-0.95 (m, 2H); MS(ESI+) for C₁₉H₂₆N₄O₄ (parent) m/z 375.3 (M+H)⁺; HPLC retention time:3.21 min (Method A).

Example 4 Scheme 1:(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-1-{[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]carbamoyl}-4-oxoazetidine-2-carboxylicacid trifluoroacetate

Step 1. Preparation of(S)-N-[(1E)-(2,2-difluoro-1,3-benzodioxol-5-yl)methylene]-2-methylpropane-2-sulfinamide

To a solution of (S)-2-methyl-propane-2-sulfinic acid amide (0.207 g,1.71 mmol) in DCM (5 mL) was added copper(II) sulfate (0.8186 g, 5.129mmol) followed by a solution of2,2-difluoro-1,3-benzodioxole-5-carbaldehyde (0.350 g, 1.88 mmol) in DCM(5 mL). The mixture was stirred at ambient temperature for 16 h,filtered through celite and concentrated at reduced pressure. Theresidue was purified by flash chromatography (hexane with ethyl acetate(2-8%) as eluent) to afford the title compound (0.468 g, 95%) as an oil.MS (ESI+) for C₁₃H₁₇F₂NO₃S m/z 290.1 (M+H)⁺; HPLC retention time: 4.73min (Method C).

Step 2. Preparation ofN-[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]-2-methylpropane-2-sulfinamide

To a cooled (−20° C.) solution of(S)-N-[(1E)-(2,2-difluoro-1,3-benzodioxol-5-yl)methylene]-2-methylpropane-2-sulfinamide(0.274 g, 0.947 mmol) in THF (9 mL) was added 3 M methylmagnesiumbromide (3 M in ethyl ether, 3.157 mL, 9.471 mmol) over 15 min at a ratesufficiently slow to maintain the reaction temperature below −15° C.After 60 min at −20° C., the reaction was quenched by the addition ofsat aqueous NH₄Cl. The mixture was diluted with ethyl acetate and washedwith sat NaHCO₃, brine, dried with anhydrous sodium sulfate, filteredand concentrated. The residue was purified by flash chromatography(hexanes with ethyl acetate 20-30% as eluent) to afford the titleproduct (the major and slower eluting diastereomer, 0.234 g, 81%) as anoil: MS (ESI+) for C₁₃H₁₇F₂NO₃S m/z 306.2 (M+H)⁺; HPLC retention time:4.20 min (Method C).

Step 3. Preparation of(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethanamine hydrochloride

To a cooled (0-5° C.) solution ofN-[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]-2-methylpropane-2-sulfinamide(0.234 g, 0.766 mmol) in methanol (4.6 mL) was added hydrogen chloridein 1,4-dioxane (4 M, 0.958 mL, 3.83 mmol). After 5 min, the ice bath wasremoved and the reaction mixture stirred at ambient temperature for 1 h.Volatiles were removed in vacuo and the residue concentrated twice frommethanol, twice from ethyl ether and dried in vacuo to afford the titlecompound (0.175 g, 96%) as a white solid: ¹H NMR (300 MHz, CD₃OD) δ1.67(t, J=7 Hz, 3H), 4.55 (q, J=7 Hz, 2H), 7.29-7.36 (m, 2H), 7.43-7.44 (m,1H); HPLC retention time: 2.69 min (Method C).

Step 4. Preparation of phenyl[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]carbamate

To a cooled (0-5° C.) solution of(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethanamine hydrochloride (100mg, 0.421 mmol) in methylene chloride (2 mL) was added triethylamine(0.132 mL, 0.947 mmol) followed by phenyl chloroformate (0.054 mL, 0.433mmol) dropwise. After 15 min the ice bath was removed and the reactionmixture stirred at ambient temperature for 3h. The mixture is dilutedwith ethyl acetate and washed with 0.1 N HCl, sat NaHCO₃, brine, driedwith anhydrous sodium sulfate, filtered and concentrated. The residuewas purified by flash chromatography (hexanes with ethyl acetate 5% aseluent) to afford the title compound (140 mg, 93%) as a white solid: MS(ESI+) for C₁₆H₁₃F₂NO₄ m/z 322.2 (M+H)⁺; HPLC retention time: 4.75 min(Method C).

Step 5. Preparation of 4-methoxybenzyl(2S,3R)-3-({2-[tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-1-{[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]carbamoyl}-4-oxoazetidine-2-carboxylate

To a solution of 4-methoxybenzyl(2S,3R)-3-({2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-4-oxoazetidine-2-carboxylate(prepared as described for Example 3 substituting 4-methoxybenzylalcohol for benzyl alcohol in step 2, 0.161 g, 0.287 mmol) in dimethylsulfoxide (2 mL) was added phenyl[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]carbamate (0.120 g,0.374 mmol). After 16 h, the mixture was diluted with ethyl acetate andwashed with 0.1 N HCl, sat NaHCO₃, brine, dried with anhydrous sodiumsulfate, filtered and concentrated. The residue was purified by flashchromatography (hexanes with ethyl acetate 5-20% as eluent) to affordthe title compound (156 mg, 69%) as a white solid: MS (ESI+) forC₄₁H₄₂F₂N₄O₁₀ m/z 789.5 (M+H)⁺; HPLC retention time: 5.71 min (MethodC).

Step 6. Preparation of(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-1-{[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]carbamoyl}-4-oxoazetidine-2-carboxylicacid trifluoroacetate

To a cooled (0-5° C.) solution of 4-methoxybenzyl(2S,3R)-3-({2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-1-{[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]carbamoyl}-4-oxoazetidine-2-carboxylate(0.160 g, 0.203 mmol) in methylene chloride (6 mL) was addedtriethylsilane (1 mL) followed by trifluoroacetic acid (3 mL). After 2h, the ice bath was removed and the reaction mixture stirred at ambienttemperature for 16 h. The volatiles were removed and the residuepurified by CombiFlash chromatography [30 g RediSep C-18 gold silica gelcartridge, solvent gradient: 10% acetonitrile (0.07% TFA)/water (0.1%TFA) to 100% acetonitrile (0.07% TFA)] and lyophilized to afford thetitle compound (80 mg, 70%) as a white solid: ¹H NMR (300 MHz, CD₃OD)δ1.54 (d, J=7 Hz, 3H), 3.20-3.29 (m, 2H), 3.69-3.75 (m, 1H), 4.30 (d,J=3 Hz, 1H), 4.94-5.00 (m, 1H), 6.90-6.98 (m, 2H), 7.13-7.26 (m, 3H); MS(ESI+) for C₂₀H₁₈F₂N₄O₆ m/z 449.2 (M+H)⁺; HPLC retention time: 3.39 min(Method C).

Example 5 Scheme 1:(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-1-{[(1S)-1-cyclohexyl-2,2,2-trifluoroethyl]carbamoyl}-4-oxoazetidine-2-carboxylicacid trifluoroacetate

Step 1. Preparation of benzyl(2S,3R)-3-({2-[tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-1-{[(1S)-1-cyclohexyl-2,2,2-trifluoroethyl]carbamoyl}-4-oxoazetidine-2-carboxylate

To a solution of benzyl(2S,3R)-3-({2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-4-oxoazetidine-2-carboxylate(100 mg, 0.19 mmol, prepared by the general methods described inScheme 1) in dimethyl sulfoxide (0.58 mL) was added phenyl[(1S)-1-cyclohexyl-2,2,2-trifluoroethyl]carbamate (74 mg, 0.24 mmol)followed by triethylamine (28 μL, 0.21 mol). The mixture was stirred atambient temperature overnight, diluted with ethyl acetate, washed withwater, dried with anhydrous sodium sulfate, filtered and concentrated.The residue was purified by flash chromatography using hexanes/ethylacetate (5-15%) as eluent to afford the title compound (70 mg, 50%) as atan solid: ¹H NMR (CDCl₃) δ0.97-1.33 (m, 5H), 1.42 (s, 9H), 1.59-1.86(m, 6H), 3.05-3.25 (m, 2H), 3.53-3.59 (m, 1H), 3.78 (s, 3H), 4.31-4.39(overlapping m, 2H), 5.09-5.25 (overlapping m, 4H), 6.64 (d, J=10 Hz,1H), 6.78-6.86 (overlapping m, 3H), 7.19-7.23 (m, 4H), 7.32-7.36 (m,3H), 7.65 (s, 1H), 8.27 (d, J=5 Hz, 1H); MS (ESI+) for C₃₉H₄₅F₃N₄O₇ m/z739.3 (M+H)^(±). HPLC retention time: 5.99 min (Method C).

Step 2. Preparation of(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-1-{[(1S)-1-cyclohexyl-2,2,2-trifluoroethyl]carbamoyl}-4-oxoazetidine-2-carboxylicacid trifluoroacetate

To a flask containing Pd/C (10%, 10 mg) was added a solution of benzyl(2S,3R)-3-({2-[tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-1-{[(1S)-1-cyclohexyl-2,2,2-trifluoroethyl]carbamoyl}-4-oxoazetidine-2-carboxylate(70 mg, 0.095 mmol) in ethanol (40 mL). The mixture was stirred under 1atmosphere of H₂ for 3 h, filtered through a pad of solka floc andconcentrated under reduced pressure. To a cooled (0-5° C.) solution ofthe residue obtained above in methylene chloride (3 mL) was addedtriethylsilane (0.5 mL) followed by trifluoroacetic acid (1.5 mL). After2 h, the ice bath was removed and the reaction mixture stirred atambient temperature for 16 h. The volatiles were removed under reducedpressure and the residue purified by CombiFlash chromatography [30 gRediSep C-18 gold silica gel cartridge, solvent gradient: 10%acetonitrile (0.07% TFA)/water (0.1% TFA) to 100% acetonitrile (0.07%TFA)] and lyophilized to afford the title compound (40 mg, 78%) as awhite solid: ¹H NMR (CDCl₃) δ1.08-1.39 (m, 5H), 1.68-1.92 (m, 6H),3.23-3.29 (m, 2H), 3.76-3.82 (m, 1H), 4.34-4.39 (overlapping m, 2H),6.90-6.93 (m, 1H), 6.98 (s, 1H), 7.78-7.80 (m, 1H); MS (ESI+) forC₁₉H₂₃F₃N₄O₄ m/z 429.1 (M+H)⁺; HPLC retention time: 3.89 min (Method C).

Example 6 Scheme 1:(2S,3R)-1-{[(1S)-1-cyclohexyl-2,2,2-trifluoroethyl]carbamoyl}-4-oxo-3-(pyridin-4-ylmethyl)azetidine-2-carboxylicacid trifluoroacetate

To a cooled (0-5° C.) solution of 4-methoxybenzyl(2S,3R)-3-[(2-chloropyridin-4-yl)methyl]-1-{[(1S)-1-cyclohexyl-2,2,2-trifluoroethyl]carbamoyl}-4-oxoazetidine-2-carboxylate(0.190 g, 0.33 mmol, prepared by the general methods described inScheme 1) in CH₂Cl₂ (6 mL) was added trifluoroacetic acid (3 mL). After5 min, the ice bath was removed and the reaction stirred at ambienttemperature for an additional 30 min. Volatiles were removed at reducedpressure and the residue concentrated twice from ether. The residue wasdissolved in ethanol (20 mL) and added to a flask containing Pd/C (10%,20 mg). Triethylamine (0.102 mL, 0.74 mmol) was added and the mixturestirred under 1 atmosphere of H₂ for 30 min. The mixture was filteredthrough celite and concentrated under reduced pressure. The residue waspurified by CombiFlash chromatography [30 g RediSep C-18 gold silica gelcartridge, solvent gradient: 10% acetonitrile (0.07% TFA)/water (0.1%TFA) to 100% acetonitrile (0.07% TFA)] and lyophilized to afford thetitle compound (39 mg, 22%) as a white solid. ¹H NMR (CD₃OD) δ1.07-1.39(m, 5H), 1.68-1.93 (m, 6H), 3.46-3.61 (m, 2H), 3.89-3.95 (m, 1H),4.36-4.44 (overlapping m, 2 H), 8.02 (d, J=7 Hz, 2H), 8.76 (d, J=7 Hz,2H); MS (ESI+) for C₁₉H₂₂F₃N₃O₄ m/z 414.0 (M+H)⁺; HPLC retention time:3.45 min (Method C).

Example 7 Scheme 2:(2S,3R)-3-[(2-Aminopyridin-4-yl)methyl]-2-cyano-4-oxo-N-[(1R)-1-phenylethyl]azetidine-1-carboxamidetrifluoroacetate

Step 1. Preparation of(2S,3R)-3-({2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylicacid

The title compound was prepared from benzyl(2S,3R)-3-({2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylateaccording to the general procedure described in step 5 of Example 3 in98% yield as a white tacky solid. MS (ESI+) for C₂₉H₄₁N₃O₆Si m/z 556.5(M+H)⁺.

Step 2. Preparation of tert-butyl(4-{[(2S,3R)-2-carbamoyl-4-oxoazetidin-3-yl]methyl}pyridin-2-yl)(4-methoxybenzyl)carbamate

A stirred solution of(2S,3R)-3-({2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylicacid (Step 1, 417 mg, 0.750 mmol) in dry DMF (3.5 mL) under nitrogen wastreated sequentially with pyridine (38 μL, 0.47 mmol),di-tert-butyldicarbonate (205 mg, 0.938 mmol) and ammonium bicarbonate(71.2 mg, 0.900 mmol), and the resulting tan mixture was stirred at rtovernight. HPLC at 24 h indicated the reaction was complete, so themixture was diluted with water (30 mL) and extracted with DCM (3×30 mL).The combined organic phase was washed with brine (15 mL), dried overNa₂SO₄ and concentrated under reduced pressure. Purification by radialchromatography [2000 micron silica gel rotor; 5-10% MeOH/DCM eluent]gave 225 mg (68%) of the title compound as a glassy solid. MS (ESI+) forC₂₃H₂₈N₄O₅ m/z 441.4 (M+H)⁺.

Step 3. Preparation of tert-butyl(4-{[(2S,3R)-2-cyano-4-oxoazetidin-3-yl]methyl}pyridin-2-yl)(4-methoxybenzyl)carbamate

A stirred solution of tert-butyl (4-{[(2S,3R)-2-carbamoyl-4-oxoazetidin-3-yl]methyl}pyridin-2-yl)(4-methoxybenzyl)carbamate (Step 2,75.0 mg, 0.170 mmol) and pyridine (33.0 μL, 0.409 mmol) in dry THF (2mL) under nitrogen was cooled in an ice-brine-MeOH bath at −10° C. andtreated with trifluoroacetic anhydride (28.8 μL, 0.204 mmol) slowlydropwise over 3 min. The resulting homogeneous mixture was allowed toslowly warm to 0° C. over 25 min, at which point HPLC indicated thereaction was essentially complete. The mixture was diluted with water(15 mL) and extracted with EtOAc (3×15 mL), and the combined organicphase was washed with brine (10 mL), dried over MgSO₄ and concentratedunder reduced pressure to give 75 mg of a clear film. This crude productwas combined with another 72 mg crude product from an additionalreaction and purified by radial chromatography [2000 micron silica gelrotor; 5% MeOH/DCM eluent] to give 119 mg (86% for combined reactions)of the title compound as a white solid. MS (ESI+) for C₂₃H₂₆N₄O₄ m/z423.4 (M+H)⁺.

Step 4. Preparation of tert-butyl(4-{[(2S,3R)-2-cyano-4-oxo-1-{[(1R)-1-phenylethyl]-carbamoyl}azetidin-3-yl]methyl}pyridin-2-yl)(4-methoxybenzyl)carbamate

The title compound was prepared from tert-butyl(4-{[(2S,3R)-2-cyano-4-oxoazetidin-3-yl]methyl}pyridin-2-yl)(4-methoxybenzyl)carbamateand [(1R)-1-isocyanatoethyl]benzene according to the general procedureof Step 4 of Example 3 in 52% as a glassy solid. MS (ESI+) forC₃₂H₃₅N₅O₅ m/z 570.5 (M+H)⁺.

Step 5. Preparation of(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-2-cyano-4-oxo-N-[(1R)-1-phenylethyl]azetidine-1-carboxamidetrifluoroacetate

The title compound was prepared from tert-butyl(4-{[(2S,3R)-2-cyano-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidin-3-yl]methyl}pyridin-2-yl)(4-methoxybenzyl)carbamateaccording to the general procedure described in Step 6 of Example 3 in51% as a white solid. ¹H NMR (400 MHz, MeOD) δ7.82 (d, J=6.8 Hz, 1H),7.38 (m, 4H), 7.29 (m, 1H), 7.09 (bd, J =7.6 Hz, 1H), 6.97 (s, 1H), 6.91(dd, J=6.8, 1.6 Hz, 1H), 5.00 (m, 1H), 4.70 (d, J=3.2 Hz, 1H), 4.17 (td,J=8.0, 3.2 Hz, 1H), 3.27 (d, J=8.0 Hz, 2H), 1.55 (d, J=6.8 Hz, 3H); MS(ESI+) for C₁₉H₁₉N₅O₂ (parent) m/z 350.3 (M+H)⁺; HPLC retention time:2.85 min (Method A).

Example 8 Scheme 2:(2S,3R)-3-[(2-Aminopyridin-4-yl)methyl]-2-cyano-N-(diphenylmethyl)-4-oxoazetidine-1-carboxamidetrifluoroacetate

Step 1. Preparation of tert-butyl[4-({(2S,3R)-2-cyano-1-[(diphenylmethyl)carbamoyl]-4-oxoazetidin-3-yl}methyl)pyridin-2-yl](4-methoxybenzyl)carbamate

The title compound was prepared from tert-butyl(4-{[(2S,3R)-2-cyano-4-oxoazetidin-3-yl]methyl}pyridin-2-yl)(4-methoxybenzyl)carbamateand 1,1′-(isocyanatomethylene)dibenzene according to the generalprocedure of step 4 of Example 3 using a 1.5 h reaction time in 84% as aglassy solid. MS (ESI+) for C₃₇H₃₇N₅O₅ m/z 632.7 (M+H)⁺.

Step 2. Preparation of(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-2-cyano-N-(diphenylmethyl)-4-oxoazetidine-1-carboxamidetrifluoroacetate

The title compound was prepared from tert-butyl[4-({(2S,3R)-2-cyano-1-[(diphenylmethyl)carbamoyl]-4-oxoazetidin-3-yl}methyl)pyridin-2-yl](4-methoxybenzyl)carbamateaccording to the general procedure of Step 6 of Example 3 in 54% as awhite solid. ¹H NMR (400 MHz, MeOD) δ7.81 (d, J=6.8 Hz, 1H), 7.44 (bd,J=7.6 Hz, 1H), 7.28-7.40 (m, 10H), 6.96 (m, 1H), 6.91 (dd, J=6.8, 1.6Hz, 1H), 6.15 (d, J=8.0 Hz, 1H), 4.73 (d, J=3.2 Hz, 1H), 4.22 (td,J=8.0, 3.2 Hz, 1H), 3.27 (d, J=8.0 Hz, 2H); MS (ESI+) for C₂₄H₂₁N₅O₂(parent) m/z 412.4 (M+H)⁺; HPLC retention time: 3.36 min (Method A).

Example 9 Scheme 3:(2S,3R)-3-[2-(2-Amino-1,3-thiazol-5-yl)ethyl]-1-[(diphenylmethyl)carbamoyl]-4-oxoazetidine-2-carboxylicacid trifluoroacetate

Step 1. Preparation of benzyl(2S,3R)-1-[tert-butyl(dimethyl)silyl]-4-oxo-3-pent-4-en-1-ylazetidine-2-carboxylate

The title compound was prepared following the general procedure of steps1 and 2 of Example 3 but using 5-bromopent-1-ene in step 1 and allowingthe alkylation to warm to 0° C. The title compound was obtained in 29%as a viscous oil. MS (ESI+) for C₂₂H₃₃NO₃Si m/z 388.3 (M+H)⁺.

Step 2. Preparation of benzyl(2S,3R)-1-[tert-butyl(dimethyl)silyl]-4-oxo-3-(4-oxobutyl)azetidine-2-carboxylate

A stirred mixture of benzyl(2S,3R)-1-[tert-butyl(dimethyl)silyl]-4-oxo-3-pent-4-en-1-ylazetidine-2-carboxylate(412 mg, 1.06 mmol) in 1,4-dioxane (12 mL)/water (3 mL) was treated withN-methylmorpholine N-oxide (156 mg, 1.33 mmol) followed by osmiumtetroxide (2.5 wt % in 2-methyl-2-propanol, 670 μL, 0.053 mmol), and theresulting mixture was stirred at rt under nitrogen for 2.5 h, at whichpoint conversion to the benzyl(2S,3R)-1-[tert-butyl(dimethyl)silyl]-3-(4,5-dihydroxypentyl)-4-oxoazetidine-2-carboxylateintermediate [MS (ESI+) for C₂₂H₃₅NO₅Si m/z 422.2 (M+H)⁺] appearedcomplete by HPLC. Sodium metaperiodate (284 mg, 1.33 mmol) was added,and the resulting heterogeneous white mixture was stirred at rt for 2.5h, at which point HPLC indicated the intermediate was consumed. Themixture was quenched with half-sat aqueous sodium thiosulfate (15 mL),diluted with water (15 mL) and extracted with EtOAc (2×40 mL). Thecombined organic phase was washed with brine (15 mL), dried over MgSO₄,filtered, concentrated and dried under reduced pressure to give 418 mg(100%) of the title compound as a viscous oil which was used withoutfurther purification. MS (ESI+) for C₂₁H₃₁NO₄Si m/z 390.3 (M+H)⁺.

Step 3. Preparation of benzyl(2S,3R)-3-[2-(2-amino-1,3-thiazol-5-yl)ethyl]-4-oxoazetidine-2-carboxylate

A stirred solution of benzyl(2S,3R)-1-[tert-butyl(dimethyl)silyl]-4-oxo-3-(4-oxobutyl)azetidine-2-carboxylate(414 mg, 1.06 mmol) in acetonitrile (11 mL) under nitrogen was treatedwith tetrabutylammonium tribromide (512 mg, 1.06 mmol) in one portion,and the resulting deep yellow, homogeneous mixture was stirred at rt for75 min., at which point HPLC indicated starting material was consumed.The mixture was diluted with water (40 mL) and extracted with EtOAc(2×40 mL), and the combined organic phase was washed with water (2×40mL) and brine (20 mL), dried over MgSO₄, filtered and concentrated underreduced pressure to give 302 mg of the crude benzyl(2S,3R)-3-(3-bromo-4-oxobutyl)-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylateintermediate [MS (ESI+) for C₂₁H₃₀BrNO₄Si m/z 468 (M+H)⁺] as a tanviscous oil. A solution of this intermediate in EtOH (11 mL) was treatedwith thiourea (89.0 mg, 1.17 mmol), and the mixture was heated at gentlereflux for 2 h, cooled to rt, diluted with DCM (45 mL), washed with sataqueous NaHCO₃ (30 mL), water (30 mL) and brine (20 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure. Purificationby silica gel chromatography [40 g, 2.5-7.5% MeOH/DCM eluent] gave 60 mg(17%) of the title compound as a tan solid. MS (ESI+) for C₁₆H₁₇N₃O₃Sm/z 332.2 (M+H)⁺. The TBS-protected by-product, benzyl(2S,3R)-3-[2-(2-amino-1,3-thiazol-5-yl)ethyl]-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylate,was also isolated (145 mg, 24%, 80% purity). MS (ESI+) for C₂₂H₃₁N₃O₃SSim/z 446.2 (M+H)⁺.

Step 4. Preparation of benzyl(2S,3R)-3-(2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}ethyl)-4-oxoazetidine-2-carboxylate

A stirred solution of benzyl(2S,3R)-3-[2-(2-amino-1,3-thiazol-5-yl)ethyl]-4-oxoazetidine-2-carboxylate(57.0 mg, 0.172 mmol) in acetonitrile (5 mL) under nitrogen was treatedwith di-tert-butyldicarbonate (48.8 mg, 0.224 mmol) and stirred at rtfor approximately 2 days, during which time additionaldi-tert-butyldicarbonate (13 mg, 0.060 mmol) was added in one portion.The mixture was concentrated under reduced pressure and purified byradial chromatography [2000 micron silica gel rotor, 2.5-10% MeOH/DCMeluent] to give 74 mg (81%) of the title compound as a glassy film(contaminated with the bis-BOC by-product benzyl(2S,3R)-3-(2-{2-[bis(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}ethyl)-4-oxoazetidine-2-carboxylate).MS (ESI+) for C₂₁H₂₅N₃O₅S m/z 432.2 (M+H)⁺.

Step 5. Preparation of benzyl(2S,3R)-3-(2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}ethyl)-1-[(diphenylmethyl)carbamoyl]-4-oxoazetidine-2-carboxylate

The title compound was prepared from(2S,3R)-3-(2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}ethyl)-4-oxoazetidine-2-carboxylateand 1,1′-(isocyanatomethylene)dibenzene following the general procedureof step 4 of Example 3 and using a 5 h reaction time in 82% as a glassysolid. MS (ESI+) for C₃₅H₃₆N₄O₆S m/z 641.3 (M+H)⁺.

Step 6. Preparation of(2S,3R)-3-(2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}ethyl)-1-[(diphenylmethyl)carbamoyl]-4-oxoazetidine-2-carboxylicacid

A mixture of benzyl(2S,3R)-3-(2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}ethyl)-1-[(diphenylmethyl)carbamoyl]-4-oxoazetidine-2-carboxylate(85.0 mg, 0.133 mmol) in 1:1 MeOH/EtOAc (6 mL) under nitrogen wastreated with 10% palladium-on-carbon (35 mg, 0.013 mmol Pd), and themixture was evacuated and filled with nitrogen twice and then stirredunder a hydrogen atmosphere (double-layer balloon) for approximately 6h, during which additional 10% palladium-on-carbon (35 mg, 0.013 mmolPd) was added in one portion. The catalyst was filtered off throughsolka floc, rinsing with 1:1 MeOH/EtOAc, and the filtrate wasconcentrated under reduced pressure to give 71 mg (97%) of the titlecompound as an off-white foam which was used without furtherpurification. MS (ESI+) for C₂₈H₃₀N₄O₆S m/z 551.2 (M+H)⁺.

Step 7. Preparation of(2S,3R)-3-[2-(2-amino-1,3-thiazol-5-yl)ethyl]-1-[(diphenylmethyl)carbamoyl]-4-oxoazetidine-2-carboxylicacid trifluoroacetate

The title compound was prepared from(2S,3R)-3-(2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}ethyl)-1-[(diphenylmethyl)carbamoyl]-4-oxoazetidine-2-carboxylicacid following the general procedure of step 6 of Example 3 and using a5 h reaction time in 47% as a white solid. ¹H NMR (400 MHz, MeOD)δ7.26-7.39 (m, 10H), 6.96 (br s, 1H), 6.11 (br s, 1H), 4.31 (br s, 1H),3.40 (m, 1H), 2.88 (m, 2H), 2.16 (m, 2H); MS (ESI+) for C₂₃H₂₂N₄O₄S(parent) m/z 451.0 (M+H)⁺; HPLC retention time: 3.25 min (Method A).

Example 10 Scheme 3:(2S,3R)-3-[2-(2-Amino-1,3-thiazol-5-yl)ethyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylicacid trifluoroacetate

Step 1. Preparation of benzyl(2S,3R)-3-(2-{2-[tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}ethyl)-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylate

The title compound was prepared from benzyl(2S,3R)-3-(2-{2-[tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}ethyl)-4-oxoazetidine-2-carboxylateand [(1R)-1-isocyanatoethyl]benzene following the general procedure ofstep 4 of Example 3 in 66% as a glassy solid. MS (ESI+) for C₃₀H₃₄N₄O₆Sm/z 579.3 (M+H)⁺.

Step 2. Preparation of(2S,3R)-3-(2-{2-[tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}ethyl)-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylicacid

The title compound was prepared from benzyl(2S,3R)-3-(2-{2-[tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}ethyl)-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylatefollowing the general procedure of step 6 of Example 9 in 82% as a whitesolid. MS (ESI+) for C₂₃H₂₈N₄O₆S m/z 489.1 (M+H)⁺.

Step 3. Preparation of(2S,3R)-3-[2-(2-amino-1,3-thiazol-5-yl)ethyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylicacid trifluoroacetate

The title compound was prepared from(2S,3R)-3-(2-{2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}ethyl)-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylicacid following the general procedure of Step 6 of Example 3 and using a5 h reaction time in 66% as a white solid. ¹H NMR (400 MHz, MeOD) δ7.36(m, 4H), 7.27 (m, 1H), 6.96 (br s, 1H), 4.98 (q, J=6.8 Hz, 1H), 4.26 (brs, 1H), 3.35 (m, 1H), 2.89 (t, J=7.2 Hz, 2H), 2.16 (m, 2H), 1.53 (d,J=6.8 Hz, 3H); MS (ESI+) for C₁₈H₂₀N₄O₄S (parent) m/z 389.1 (M+H)⁺; HPLCretention time: 2.71 min (Method A).

Example 11 Scheme 4:(2S,3R)-3-[(2-Amino-1,3-thiazol-5-yl)methyl]-1-[(diphenylmethyl)carbamoyl]-4-oxoazetidine-2-carboxylicacid trifluoroacetate

Step 1. Preparation of benzyl(2S,3R)-1-[tert-butyl(dimethyl)silyl]-3-[(2E)-3-chloroprop-2-en-1-yl]-4-oxoazetidine-2-carboxylate

The title compound was prepared following the general procedures ofsteps 1-2 of Example 3 but using (1E)-1,3-dichloroprop-1-ene fortert-butyl [4-(bromomethyl)pyridin-2-yl](4-methoxybenzyl)carbamate instep 1 and allowing the reaction temperature to warm to 0° C. The titlecompound was obtained in 39% (mixture of E/Z isomers) as a viscous oil.MS (ESI+) for C₂₀H₂₈ClNO₃Si m/z 394.2 (M+H)⁺.

Step 2. Preparation of benzyl(2S,3R)-3-[(2-amino-1,3-thiazol-5-yl)methyl]-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylate

A stirred solution of benzyl(2S,3R)-1-[tert-butyl(dimethyl)silyl]-3-[(2E)-3-chloroprop-2-en-1-yl]-4-oxoazetidine-2-carboxylate(220 mg, 0.558 mmol) in dry 1,2-dichloroethane (2.8 mL) in a screw-capvial was treated with m-chloroperbenzoic acid (289 mg, 1.68 mmol, washedand dried, >90% purity) and 2,6-di-tert-butyl-4-methylphenol (7.0 mg,0.032 mmol), heated quickly to 60° C. and stirred at this temperaturefor 18 h. The mixture was cooled to rt, diluted with EtOAc (20 mL),washed with sat aqueous NaHCO₃ (2×20 mL), half-sat aqueous sodiumthiosulfate (2×15 mL), water (20 mL) and brine (10 mL), dried over MgSO₄and concentrated under reduced pressure to give the crude benzyl(2S,3R)-1-[tert-butyl(dimethyl)silyl]-3-[(3-chlorooxiran-2-yl)methyl]-4-oxoazetidine-2-carboxylateintermediate [MS (ESI+) for C₂₀H₂₈ClNO₄Si m/z 410.2 (M+H)⁺] as a viscousoil which was used without further purification. A stirred mixture ofthis intermediate in dry 1,2-dimethoxyethane (5.5 mL) under nitrogen wastreated with thiourea (53.1 mg, 0.698 mmol), heated to 60° C. andstirred at this temperature for 2.5 h, at which point HPLC and LC-MSindicated the intermediate was consumed. The mixture was cooled to rt,diluted with EtOAc (30 mL), water (8 mL) and sat aqueous NaHCO₃ (8 mL),and the layers were separated. The organic phase was washed with sataqueous NaHCO₃ (15 mL), water (15 mL) and brine (10 mL), dried overMgSO₄ and concentrated under reduced pressure to give 240 mg of thecrude product, which was combined with 100 mg crude product from aprevious reaction. Purification by radial chromatography [2000 micronsilica gel rotor; 5-10% MeOH/DCM eluent] gave 172 mg (49% for combinedreactions) of the title compound as a viscous oil. MS (ESI+) forC₂₁H₂₉N₃O₃SSi m/z 432.3 (M+H)⁺.

Step 3. Preparation of benzyl(2S,3R)-3-({2-[tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}methyl)-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylate

A stirred solution of benzyl(2S,3R)-3-[(2-amino-1,3-thiazol-5-yl)methyl]-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylate(168 mg, 0.389 mmol) in acetonitrile (6 mL) under nitrogen was treatedwith di-tert-butyldicarbonate (170 mg, 0.778 mmol), and the resultingmixture was stirred at rt for approx. 24 h, at which point HPLCindicated reaction was complete. The mixture was concentrated underreduced pressure and purified by radial chromatography [2000 micronsilica gel rotor; 30-50% EtOAc/hexanes eluent] to give the 242 mg (100%)of the title compound as an oily film (contaminated with the bis-BOCby-product benzyl(2S,3R)-3-({2-[bis(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}methyl)-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylate).MS (ESI+) for C ₂₆H₃₇ N₃O₅ S S i m/z 532.3 (M+H)⁺.

Step 4. Preparation of benzyl(2S,3R)-3-({2-[tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}methyl)-4-oxoazetidine-2-carboxylate

The title compound was prepared from benzyl(2S,3R)-3-({2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}methyl)-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylatefollowing the general procedure of step 3 of Example 3 in 99%(contaminated with bis-BOC by-product benzyl(2S,3R)-3-({2-[bis(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}methyl)-4-oxoazetidine-2-carboxylate).MS (ESI+) for C₂₀H₂₃N₃O₅S m/z 418.2 (M+H)⁺.

Step 5. Preparation of benzyl(2S,3R)-3-({2-[tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}methyl)-1-[(diphenylmethyl)carbamoyl]-4-oxoazetidine-2-carboxylate

The title compound was prepared from benzyl(2S,3R)-3-({2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}methyl)-4-oxoazetidine-2-carboxylateand 1,1′-(isocyanatomethylene)dibenzene following the general procedureof step 4 of Example 3 using a 3 h reaction time in 88% as a glassysolid. MS (ESI+) for C₃₄H₃₄N₄O₆S m/z 627.4 (M+H)⁺.

Step 6. Preparation of(2S,3R)-3-({2-[tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}methyl)-1-[diphenylmethyl)carbamoyl]-4-oxoazetidine-2-carboxylicacid

The title compound was prepared from benzyl(2S,3R)-3-({2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}methyl)-1-[(diphenylmethyl)carbamoyl]-4-oxoazetidine-2-carboxylatefollowing the general procedure of step 6 of Example 9 in quantitativeyield as a white solid. MS (ESI+) for C₂₇H₂₈N₄O₆S m/z 537.3 (M+H)⁺.

Step 7. Preparation of(2S,3R)-3-[(2-amino-1,3-thiazol-5-yl)methyl]-1-[(diphenylmethyl)carbamoyl]-4-oxoazetidine-2-carboxylicacid trifluoroacetate

The title compound was prepared from(2S,3R)-3-({2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}methyl)-1-[(diphenylmethyl)carbamoyl]-4-oxoazetidine-2-carboxylicacid following the general procedure of step 6 of Example 3 and using a3 h reaction time in 53% as a white solid. ¹H NMR (400 MHz, MeOD) δ7.49(d, J=8.4 Hz, 1H), 7.40-7.27 (m, 10H), 7.14 (s, 1H), 6.12 (m, 1H), 4.34(d, J=2.8 Hz, 1H), 3.69 (td, J=7.2, 2.8 Hz, 1H), 3.25 (m, 2H); MS (ESI+)for C₂₂H₂₀N₄O₄S (parent) m/z 437.1 (M+H)⁺; HPLC retention time: 3.11 min(Method A).

Example 12 Scheme 4:(2S,3R)-3-[(2-Amino-1,3-thiazol-5-yl)methyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylicacid trifluoroacetate

Step 1. Preparation of benzyl(2S,3R)-3-({2-[tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}methyl)-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylate

The title compound was prepared from benzyl(2S,3R)-3-({2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}methyl)-4-oxoazetidine-2-carboxylateand [(1R)-1-isocyanatoethyl]benzene following the general procedure ofstep 4 of Example 3, in approx. 95% as a glassy solid. MS (ESI+) forC₂₉H₃₂N₄O₆S m/z 565.2 (M+H)⁺.

Step 2. Preparation of(2S,3R)-3-({2-[tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}methyl)-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylicacid

The title compound was prepared from benzyl(2S,3R)-3-({2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}methyl)-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylatefollowing the general procedure of step 6 of Example 9 in 75% as a whitesolid. MS (ESI+) for C₂₂H₂₆N₄O₆S m/z 475.2 (M+H)⁺.

Step 3. Preparation of(2S,3R)-3-[(2-amino-1,3-thiazol-5-yl)methyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylicacid trifluoroacetate

The title compound was prepared from(2S,3R)-3-({2-[(tert-butoxycarbonyl)amino]-1,3-thiazol-5-yl}methyl)-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylicacid following the general procedure of step 6 of Example 3 using a 5 hreaction time in 54% as a white solid. ¹H NMR (400 MHz, MeOD) δ7.37 (m,4H), 7.28 (m, 1H), 7.16 (s, 1H), 4.98 (q, J=6.8 Hz, 1H), 4.32 (d, J=2.8Hz, 1H) 3.65 (td, J=7.2, 2.8 Hz, 1H), 3.25 (m, 2H), 1.54 (d, J=7.2H,3H); MS (ESI+) for C₁₇H₁₈N₄O₄S (parent) m/z 375.3 (M+H)⁺; HPLC retentiontime: 2.58 min (Method A).

Example 13 Scheme 5:(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-4-oxo-N-1-[(1R)-1-phenylethyl]azetidine-1,2-dicarboxamidetrifluoroacetate

Step 1. Preparation of di-tert-butyl[4-({(3R,4S)-1-[tert-butyl(dimethyl)silyl]-2-oxo-4-[(2,4,6-trimethoxybenzyl)carbamoyl]azetidin-3-yl}methyl)pyridin-2-yl]imidodicarbonate

A solution of(2S,3R)-3-({2-[bis(tert-butoxycarbonyl)amino]pyridin-4-yl}methyl)-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylicacid (170 mg, 0.32 mmol) in N,N-dimethylformamide (2.0 mL) was treatedwith (2,4,6-trimethoxyphenyl)methanamine hydrochloride (81.6 mg, 0.349mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (145 mg, 0.381 mmol) followed by dropwise additionof N,N-diisopropylethylamine (182 μL, 1.05 mmol), and the reactionmixture was stirred at 0° C. HPLC analysis after 15 min indicated thestarting material had been consumed. The reaction mixture was dilutedwith 25 mL H₂O, extracted with two 25 mL portions of CH₂Cl₂ and thecombined organic phase was washed with 20 mL portions of H₂O and brine.The organic phase was dried over Na₂SO₄, filtered and concentrated toyield a slightly tan liquid that was placed on high vac to remove DMF.The crude material was purified by flash chromatography (60 g silicagel, 15-50% EtOAc/CH₂Cl₂) to yield the title compound (130 mg, 60%) as aslightly yellow glass: HPLC retention time: 5.05 min (Method A); MS(ESI+) for C₃₆H₅₄N₄O₉Si m/z 715.5 (M+H)⁺.

Step 2. Preparation of di-tert-butyl[4-({(3R,4S)-2-oxo-4-[(2,4,6-trimethoxybenzyl)carbamoyl]azetidin-3-yl}methyl)pyridin-2-yl]imidodicarbonate

A solution of di-tert-butyl[4-({(3R,4S)-1-[tert-butyl(dimethyl)silyl]-2-oxo-4-[(2,4,6-trimethoxybenzyl)carbamoyl]azetidin-3-yl}methyl)pyridin-2-yl]imidodicarbonate(137 mg, 0.192 mmol) in methanol (3.3 mL) was treated dropwise withacetic acid (38 μL, 0.67 mmol) followed by 0.5 M ammonium fluoride inmethanol (0.46 mL, 0.23 mmol) and the reaction mixture was stirred atroom temperature. HPLC after 1.5 h indicated the reaction was complete.The reaction mixture was concentrated, the residue diluted with 7 mLtoluene and concentrated. The residue was taken up in 20 mL CH₂Cl₂ andwashed with 10 mL portions of H₂O and sat NaHCO₃. The organic phase wasdried over Na₂SO₄, filtered and concentrated to yield the title compound(70 mg, 95%) as a light yellow stiff foam: HPLC retention time, 3.84 min(Method A); MS (ESI+) for C₃₀H₄₀N₄O₉ m/z 601.3 (M+H)⁺.

Step 3. Preparation of di-tert-butyl[-4-({(3R,4S)-2-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}-4-[(2,4,6-trimethoxybenzyl)carbamoyl]azetidin-3-yl}methyl)pyridin-2-yl]imidodicarbonate

A solution of di-tert-butyl[4-({(3R,4S)-2-oxo-4-[(2,4,6-trimethoxybenzyl)carbamoyl]azetidin-3-yl}methyl)pyridin-2-yl]imidodicarbonate(94 mg, 0.16 mmol) in methylene chloride (2.5 mL) was treated dropwisewith triethylamine (87 μL, 0.63 mmol) followed by[(1R)-1-isocyanatoethyl]benzene (29 μL, 0.20 mmol) and the reactionmixture was stirred at room temperature. After 4 h, HPLC indicated thereaction was complete. The reaction mixture was concentrated and theresidue was taken up in 5 mL CH₂Cl₂ and concentrated to a slightlyyellow glass. The crude material was purified by flash chromatography(30 g silica gel, 40-70% EtOAc/hex) to yield the title compound (94 mg,80%) as a colorless glass: HPLC retention time, 4.88 min (Method A); MS(ESI+) for C₃₉H₄₉N₅O₁₀ m/z 748.9 (M+H)⁺; MS (ESI−) for C₃₉H₄₉N₅O₁₀ m/z746.5 (M−H)⁻.

Step 4. Preparation of(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-4-oxo-N-1-[(1R)-1-phenylethyl]azetidine-1,2-dicarboxamidetrifluoroacetate

A solution of di-tert-butyl[4-({(3R,4S)-2-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}-4-[(2,4,6-trimethoxybenzyl)carbamoyl]azetidin-3-yl}methyl)pyridin-2-yl]imidodicarbonate(94 mg, 0.12 mmol) in methylene chloride (4.0 mL) was cooled at 0° C.,treated dropwise with trifluoroacetic acid (1.0 mL, 13 mmol) and themixture was stirred at 0° C. for 60 minutes at which time the reactionwas allowed to warm to room temperature. After stirring for 24 h, thereaction was found to be complete by HPLC. The reaction mixture wasconcentrated and the crude material was purified by CombiFlashchromatography [30 g RediSep C-18 gold silica gel cartridge, solventgradient: 10% acetonitrile (0.07% TFA)/water (0.1% TFA) to 100%acetonitrile (0.07% TFA)] to yield the title compound (31 mg, 51%) as awhite solid after lyophilization: HPLC retention time: 2.58 min (MethodA); MS (ESI+) for C₁₉H₂₁N₅O₃ m/z 368.1 (M+H)⁺; ¹H NMR (400 MHz, MeOD)δ7.96 (1H, br. s.) 7.77 (1H, d, J=6.6 Hz) 7.40 (1H, br. s.) 7.35 (4H, m)7.26 (1H, m) 7.09 (1H, d, J=7.8 Hz) 6.96 (1H, s) 6.89 (1H, dd, J=6.7,1.6 Hz) 4.96 (1H, m) 4.28 (1H, d, J=2.8 Hz) 3.67 (1H, dt, J=8.0, 2.8 Hz)3.23 (2H, m) 1.52 (3H, d, J=7.1 Hz).

Example 14 Scheme 6:4-{[(2R,3R)-3-[(2-aminopyridin-4-yl)methyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidin-2-yl]oxy}benzoicacid trifluoroacetate

Step 1. Preparation of(2R,3R)-3-({2-[bis(tert-butoxycarbonyl)amino]pyridin-4-yl}methyl)-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidin-2-yl3-chlorobenzoate

A solution of(2S,3R)-3-({2-[bis(tert-butoxycarbonyl)amino]pyridin-4-yl}methyl)-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidine-2-carboxylicacid (201 mg, 0.375 mmol) in methylene chloride (7.0 mL) was cooled at0° C. with an ice bath and treated with m-chloroperbenzoic acid (79 mg,0.41 mmol) followed by N,N′-dicyclohexylcarbodiimide (85 mg, 0.41 mmol)and the reaction mixture was stirred at 0° C. for 25 min at which pointHPLC indicated the starting material had been consumed. The reactionmixture was filtered, the solid was washed with 20 mL of 1/1 Et₂O/CH₂Cl₂and the filtrate was washed with 15 mL H₂O and brine. The organic phasewas dried over Na₂SO₄, filtered and concentrated to yield a light yellowglass. The crude material was purified by flash chromatography (45 gsilica gel, 10-30% EtOAc/hex) to yield the title compound (131 mg, 54%)as a colorless glass: HPLC retention time, 5.95 min (Method A); MS(ESI+) for C₃₂H₄₄ClN₃O₇Si m/z 646.3/648.3 (M+H)⁺.

Step 2. Preparation of(2R,3R)-3-({2-[bis(tert-butoxycarbonyl)amino]pyridin-4-yl}methyl)-4-oxoazetidin-2-yl3-chlorobenzoate

A solution of(2R,3R)-3-({2-[bis(tert-butoxycarbonyl)amino]pyridin-4-yl}methyl)-1-[tert-butyl(dimethyl)silyl]-4-oxoazetidin-2-yl3-chlorobenzoate (130.0 mg, 0.201 mmol) in methanol (3.0 mL, 74 mmol)was treated with acetic acid (40 μL, 0.70 mmol) followed by 0.5 Mammonium fluoride in methanol (0.48 mL, 0.24 mmol) and the reactionmixture was stirred at room temperature for 1 h at which point HPLCindicated the starting material had been consumed. The reaction mixturewas concentrated in vacuo, the residue was taken up in 5 mL toluene andthe solution was concentrated. This process was repeated once, theresidue was taken up in 25 mL CH₂Cl₂ and the solution was washed with 15mL H₂O and sat. NaHCO₃ solution. The organic phase was dried overNa₂SO₄, filtered and concentrated to yield the title compound (106 mg,96%) as a slightly yellow glass: HPLC retention time: 4.43 min (MethodA); MS (ESI+) for C₂₆H₃₀ClN₃O₇ m/z 532.1/534.2 (M+H)⁺.

Step 3. Preparation of benzyl4-{[(2R,3R)-3-({2-[bis(tert-butoxycarbonyl)amino]pyridin-4-yl}methyl)-4-oxoazetidin-2-yl]oxy}benzoate

A solution of(2R,3R)-3-({2-[bis(tert-butoxycarbonyl)amino]pyridin-4-yl}methyl)-4-oxoazetidin-2-yl3-chlorobenzoate (105 mg, 0.197 mmol) in acetonitrile (13 mL) and water(1 mL) was treated with benzyl p-hydroxybenzoate (45 mg, 0.20 mmol)followed by cesium carbonate (96 mg, 0.30 mmol) and the mixture wasstirred at room temperature for 70 min, at which point HPLC indicatedthe starting material had been consumed. The reaction mixture wasdiluted with 25 mL H₂O and extracted with three 25 mL portions of EtOAc.The organic phase was washed with 25 mL brine and dried over Na₂SO₄. Thesolution was filtered and concentrated to yield a colorless glass whichwas purified by flash chromatography (30 g silica gel, 50-60% EtOAc/hex)to yield the title compound (85 mg, 71%) as a colorless glass: HPLCretention time, 4.71 min (Method A); MS (ESI+) for C₃₃H₃₇N₃O₈ m/z 604.3(M+H)⁺; MS (ESI−) for C₃₃H₃₇N₃O₈ m/z 604.6 (M−H)⁻.

Step 4. Preparation of benzyl4-{[(2R,3R)-3-({2-[bis(tert-butoxycarbonyl)amino]pyridin-4-yl}methyl)-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidin-2-yl]oxy}benzoate

A solution of benzyl4-{[(2R,3R)-3-({2-[bis(tert-butoxycarbonyl)amino]pyridin-4-yl}methyl)-4-oxoazetidin-2-yl]oxy}benzoate(82 mg, 0.14 mmol) in methylene chloride (2.2 mL) was treated dropwisewith triethylamine (76 μL, 0.54 mmol) followed by[(1R)-1-isocyanatoethyl]benzene (25 μL, 0.18 mmol) and the reactionmixture was stirred at room temperature for 4 h, at which point HPLCindicated nearly all the starting material had been consumed. Anadditional 10 μL of isocyanate was added, and the reaction was continuedfor another hour. The reaction mixture was concentrated, the residuetaken up in 5 mL CH₂Cl₂ and the solution concentrated to yield a whitestiff foam. The crude material was purified by flash chromatography (25g silica gel, 25-45 EtOAc/hex) to yield the title compound (87 mg, 85%)as a colorless glass: HPLC retention time: 5.54 min (Method A); MS(ESI+) for C₄₂H₄₆N₄O m/z 751.4 (M+H)⁺.

Step 5. Preparation of4-{[(2R,3R)-3-({2-[bis(tert-butoxycarbonyl)amino]pyridin-4-yl}methyl)-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidin-2-yl]oxy}benzoicacid

A solution of benzyl4-{[(2R,3R)-3-({2-[bis(tert-butoxycarbonyl)amino]pyridin-4-yl}methyl)-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidin-2-yl]oxy}benzoate(87 mg, 0.12 mmol) in methanol (3.0 mL) and ethyl acetate (3.0 mL) wascarefully treated with 10% palladium on carbon (12 mg). The reactionflask was evacuated and filled with hydrogen gas three times and themixture was stirred under an atmosphere of hydrogen for 4.5 h at whichpoint TLC had indicated the starting material had been consumed. Thereaction mixture was filtered through a pad of solka floc and the padwas washed with 40 mL 1/1 EtOAc/MeOH. The filtrate was concentrated toyield the title compound (77 mg, 100%) as a colorless glass: HPLCretention time, 4.56 min (Method A); MS (ESI+) for C₃₅H₄₀N₄O₉ m/z 661.3(M+H)⁺; MS (ESI−) for C₃₅H₄₀N₄O₉ m/z 559.8 (M−H)⁻.

Step 6. Preparation of4-{[(2R,3R)-3-[(2-aminopyridin-4-yl)methyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidin-2-yl]oxy}benzoicacid trifluoroacetate

A solution of4-{[(2R,3R)-3-({2-[bis(tert-butoxycarbonyl)amino]pyridin-4-yl}methyl)-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidin-2-yl]oxy}benzoicacid (76 mg, 0.12 mmol) in methylene chloride (3.3 mL) was cooled at 0°C. and treated dropwise with trifluoroacetic acid (0.66 mL, 8.6 mmol).The reaction mixture was stirred at 0° C. for 1 h, after which the icebath was allowed to slowly expire. After 7 h, HPLC indicated thestarting material had been consumed. The reaction mixture wasconcentrated in vacuo. The residue was taken up in 5 mL CH₂Cl₂ andconcentrated to yield the crude product as a light pink glass. Thematerial was purified by CombiFlash chromatography [30 g RediSep C-18gold silica gel cartridge, solvent gradient: 10% acetonitrile (0.07%TFA)/water (0.1% TFA) to 100% acetonitrile (0.07% TFA)] to yield thetitle compound (32 mg, 48%) as a white powder: HPLC retention time: 3.13min (Method A); MS (ESI+) for C₂₅H₂₄N₄O₅ m/z 461.1 (M+H)⁺; ¹H NMR (400MHz, MeOD) δ7.94 (2H, m) 7.72 (1H, d, J=6.6 Hz) 7.34 (4H, m) 7.27 (2H,m) 7.13 (2H, m) 6.91 (1H, s) 6.86 (1H, dd, J=6.8, 1.5 Hz) 6.08 (1H, d,J=1.5 Hz) 4.96 (1H, m) 3.79 (1H, dt, J=8.2, 1.5 Hz) 3.24 (2H, d, J=8.1Hz) 1.53 (3H, d, J=7.1 Hz).

Example 15 Scheme 7:(2R,3S)-3-[(2-aminopyridin-4-yl)methyl]-2-(methylsulfonyl)-4-oxo-N-[(1R)-1-phenylethyl]azetidine-1-carboxamidetrifluoroacetate

Step 1. Preparation of tert-butyl(4-methoxybenzyl)(4-{[(2R,3S)-2-(methylsulfonyl)-4-oxoazetidin-3-yl]methyl}pyridin-2-yl)carbamate

A solution of(2R,3R)-3-({2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-4-oxoazetidin-2-yl3-chlorobenzoate (100 mg, 0.181 mmol) in acetonitrile (4.0 mL) and water(1.1 mL) was treated with sodium methanesulfinate (87 mg, 0.72 mmol).The mixture was stirred at room temperature for 4 h at which time HPLCindicated the starting material had been consumed. The reaction mixturewas concentrated to remove most of the CH₃CN and diluted with 10 mL H₂O.The aqueous phase was extracted with three 10 mL portions of EtOAc. Thecombined organic phase was dried over MgSO₄, filtered and concentratedto yield a colorless glass. The crude material was purified by prep TLC(20 cm×10 cm×1.0 mm prep TLC plate, 65% EtOAc/hex) to yield the titlecompound (52 mg, 60%) as a colorless glass: HPLC retention time, 3.50min (Method A); MS (ESI+) for C₂₃H₂₉N₃O₆S m/z 476.4 (M+H)⁺; MS (ESI−)for C₂₃H₂₉N₃O₆S m/z 474.3 (M−H)⁻.

Step 2. Preparation of tert-butyl(4-methoxybenzyl)(4-{[(2R,3S)-2-(methylsulfonyl)-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidin-3-yl]methyl}pyridin-2-yl)carbamate

A solution of tert-butyl(4-methoxybenzyl)(4-{[(2R,3S)-2-(methylsulfonyl)-4-oxoazetidin-3-yl]methyl}pyridin-2-yl)carbamate(52.0 mg, 0.109 mmol) in methylene chloride (2.4 mL) was treateddropwise with triethylamine (61 μL, 0.44 mmol) followed by[(1R)-1-isocyanatoethyl]benzene (20 μL, 0.14 mmol) and the reactionmixture was stirred at room temperature for 2 h at which point HPLCindicated the starting material was consumed. The reaction mixture wasconcentrated to a colorless oil. The material was taken up in 5 mLCH₂Cl₂ and concentrated and the resultant crude product was purified byprep TLC (20 cm×20 cm×1.0 mm prep TLC plate, 50% EtOAc/hex) to yield thetitle compound (63 mg, 91%) as a colorless stiff foam: HPLC retentiontime: 5.02 min (Method A); MS (ESI+) for C₃₂H₃₈N₄O₇S m/z 623.5 (M+H)⁺;MS (ESI+) for C₃₂H₃₈N₄O₇S m/z 621.5 (M+H)⁺.

Step 3. Preparation of(2R,3S)-3-[(2-aminopyridin-4-yl)methyl]-2-(methylsulfonyl)-4-oxo-N-[(1R)-1-phenylethyl]azetidine-1-carboxamidetrifluoroacetate

A solution of tert-butyl(4-methoxybenzyl)(4-{[(2R,3S)-2-(methylsulfonyl)-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidin-3-yl]methyl}pyridin-2-yl)carbamate(62 mg, 0.10 mmol) in methylene chloride (3 mL) was cooled at 0° C. andtreated with trifluoroacetic acid (1.0 mL, 13 mmol). The solution wasstirred at 0° C. for 30 minutes then allowed to warm to roomtemperature. HPLC of the reaction mixture after 23 h indicated thestarting material had been consumed. The crude reaction mixture wasconcentrated. The residue was taken up in 5 mL CH₂Cl₂ and concentratedto yield the crude product as a pink glass. The crude material waspurified by CombiFlash chromatography [30 g RediSep C-18 gold silica gelcartridge, solvent gradient: 10% acetonitrile (0.07% TFA)/water (0.1%TFA) to 100% acetonitrile (0.07% TFA)] to yield the title compound (31mg, 60%) as a white solid after lyophilization: HPLC retention time:2.99 min (Method A); MS (ESI+) for C₁₉H₂₂N₄O₄S m/z 403.2 (M+H)⁺; ¹H NMR(400 MHz, MeOD) δ7.77 (1H, d, J=6.8 Hz) 7.35 (4H, d, J=4.3 Hz) 7.27 (1H,m) 7.19 (1H, d, J=7.6 Hz) 6.98 (1H, s) 6.91 (1H, dd, J=6.8, 1.5 Hz) 5.30(1H, d, J=2.8 Hz) 4.97 (1H, m) 4.02 (1H, m) 3.34 (1H, m) 3.21 (1H, m)3.17 (3H, s) 1.54 (3H, d, J=7.1 Hz).

Example 16 Scheme 8:(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-2-[(methoxyimino)methyl]-4-oxo-N-[(1R)-1-phenylethyl]azetidine-1-carboxamidetrifluoroacetate

Step 1. Preparation of(4S)-1-[tert-butyl(diphenyl)silyl]-4-(hydroxymethyl)azetidin-2-one

A solution of benzyl(2S)-1-[tert-butyl(diphenyl)silyl]-4-oxoazetidine-2-carboxylate (2.20 g,4.96 mmol) in methanol (20 mL) was cooled to 0° C. (ice water) andtreated with sodium tetrahydroborate (0.55 g, 15 mmol) in one portion.The reaction mixture was stirred and slowly warmed to room temperature.After 18 h, TLC indicated the starting material had been consumed. Thereaction mixture was quenched with 10 mL sat NaHCO₃ solution and themixture was concentrated to remove the solvent. The resultant milkysuspension was diluted with 20 mL H₂O and extracted with three 25 mLportions of MTBE. The organic phase was washed with 20 mL brine anddried over MgSO₄. The organic phase was filtered and concentrated toyield a colorless viscous oil. The crude material was purified by flashchromatography (110 g, 15-50% EtOAc/hex) to yield the title compound(0.82 g, 48%) as a white solid: HPLC retention time: 4.47 min (MethodA).

Step 2. Preparation of(2S)-1-[tert-butyl(diphenyl)silyl]-4-oxoazetidine-2-carbaldehyde

A solution of(4S)-1-[tert-butyl(diphenyl)silyl]-4-(hydroxymethyl)azetidin-2-one (530mg, 1.56 mmol) in methylene chloride (39 mL) was treated withDess-Martin periodinane (1.32 g, 3.12 mmol) and the reaction mixture wasstirred at room temperature producing a clear solution. After 1.5 h atroom temperature, the reaction mixture was diluted with 40 mL Et₂O andconcentrated in vacuo to afford a clear oil, which was taken up in 50 mLEt₂O and a 1/1 mixture of 40 mL 10% aqueous Na₂S₂O₃ and 40 mL sat NaHCO₃was added. The mixture was stirred vigorously until all solids haddissolved. The separated aqueous layer was extracted with two 20 mLportions of Et₂O and the combined organic phase was dried over Na₂SO₄,filtered, and concentrated to afford the title compound (520 mg 99%) asa colorless viscous oil that was used as is in the next step: ¹H NMR(300 MHz, CDCl₃) δ9.12 (1H, d, J=4.1 Hz) 7.68 (2H, m) 7.59 (2H, m) 7.45(6H, m) 3.76 (1H, m) 3.39 (1H, dd, J=15.8, 6.3 Hz) 3.02 (1H, dd, J=15.8,3.1 Hz) 1.25 (9H, s).

Step 3. Preparation of(2S)-1-[tert-butyl(diphenyl)silyl]-4-oxoazetidine-2-carbaldehydeO-methyloxime

(2S)-1-[tert-butyl(diphenyl)silyl]-4-oxoazetidine-2-carbaldehyde (520mg, 1.54 mmol) was taken up in ethanol (14 mL) and treated with pyridine(177 μL, 2.19 mmol) followed by methoxyamine hydrochloride (154 mg, 1.87mmol) in one portion. The solution was stirred at room temperature for19 h, at which point TLC (30% EA/hex) indicated the reaction wascomplete. The reaction mixture was concentrated to remove most of thesolvent and the resultant colorless liquid was diluted with 20 mL H₂Oand extracted with three 20 mL portions of MTBE. The organic phase waswashed with 20 mL H₂O, dried over Na₂SO₄, filtered and concentrated toyield a colorless viscous oil. The crude material was purified by flashchromatography (50 g, 10-20% EtOAc/hex) to yield the title compound (469mg, 82%) as a colorless viscous oil which was a mixture of E/Z geometricisomers: HPLC retention time: 4.93 min (Method A); MS (ESI+) forC₂₁H₂₆N₂O₂Si m/z 367.4 (M+H)⁺, m/z 389.3 (M+Na)⁺.

Step 4. Preparation of tert-butyl[4-({(2S,3R)-1-[tert-butyl(diphenyl)silyl]-2-[(methoxyimino)methyl]-4-oxoazetidin-3-yl}methyl)pyridin-2-yl](4-methoxybenzyl)carbamate

A solution of(2S)-1-[tert-butyl(diphenyl)silyl]-4-oxoazetidine-2-carbaldehydeO-methyloxime (469 mg, 1.28 mmol) in tetrahydrofuran (9.6 mL) was cooledto −78° C. and treated dropwise with a 1.2 M solution of lithiumdiisopropylamide in hexanes/THF/ethylbenzene (1.2 mL, 1.4 mmol). Thepale yellow reaction mixture was stirred for 15 min and transferred viacannula to a precooled (−78° C.) solution of tert-butyl[4-(bromomethyl)pyridin-2-yl](4-methoxybenzyl)carbamate (570 mg, 1.4mmol) in tetrahydrofuran (9.6 mL) dropwise over 15 min to afford ayellow-brown solution. The reaction mixture was stirred for 90 min at−78° C., at which time HPLC indicated the starting material had beenconsumed. The reaction was quenched by the addition of 10 mL saturatedaqueous NH₄Cl. The cooling bath was removed and the mixture was stirredfor 5 min. The reaction mixture was diluted with 20 mL H₂O and extractedwith two 40 mL portions of EtOAc. The combined organic phase was washedwith 20 mL portions of H₂O and brine and dried over MgSO₄. The solutionwas filtered and concentrated to yield a maroon viscous oil. The crudematerial was purified by flash chromatography (80 g silica gel, 10-30%EtOAc/hex) to yield the title compound (553 mg, 62%) as a light yellowstiff foam which was a mixture of E/Z geometric isomers: HPLC retentiontime: 5.77/5.82 min (Method A); MS (ESI+) for C₄₀H₄₈N₄O₅Si m/z 693.6(M+H)⁺.

Step 5. Preparation of tert-butyl(4-methoxybenzyl)[4-({(2S,3R)-2-[(methoxyimino)methyl]-4-oxoazetidin-3-yl}methyl)pyridin-2-yl]carbamate

A solution of tert-butyl[4-({(2S,3R)-1-[tert-butyl(diphenyl)silyl]-2-[methoxyimino)methyl]-4-oxoazetidin-3-yl}methyl)pyridin-2-yl](4-methoxybenzyl)carbamate(553 mg, 0.678 mmol) in methanol (10 mL) was treated dropwise withacetic acid (130 μL, 2.4 mmol) followed by 0.5 M ammonium fluoride inmethanol (1.6 mL, 0.81 mmol). The solution was stirred at roomtemperature for 1 h, at which time HPLC indicated the starting materialhad been consumed. The reaction mixture was concentrated to remove theMeOH and the resultant oil was taken up in 50 mL CH₂Cl₂. The organicphase was washed with 25 mL portions of sat NaHCO₃ and H₂O, dried overNa₂SO₄, filtered and concentrated to a light yellow oil. The crudematerial was purified by flash chromatography (60 g silica gel, 40-80%EtOAc/hex) to yield the title compound (250 mg, 81%) as a slightlyyellow glass which was a mixture of E/Z isomers: HPLC retention time:3.59/3.66 min (Method A); MS (ESI+) for C₂₄H₃₀N₄O₅ m/z 455.3 (M+H)⁺; MS(ESI−) for C₂₄H₃₀N₄O₅ m/z 453.3 (M−H)⁻.

Step 6. Preparation of tert-butyl(4-methoxybenzyl)(4-{[(2S,3R)-2-[(methoxyimino)methyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidin-3-yl]methyl}pyridin-2-yl)carbamate

A solution of tert-butyl(4-methoxybenzyl)[4-({(2S,3R)-2-[(methoxyimino)methyl]-4-oxoazetidin-3-yl}methyl)pyridin-2-yl]carbamate(131 mg, 0.288 mmol) in methylene chloride (4.6 mL) was treated dropwisewith triethylamine (160 μL, 1.2 mmol) followed by[(1R)-1-isocyanatoethyl]benzene (53 μL, 0.37 mmol) and the reactionmixture was stirred at room temperature for 21 h, at which point HPLCindicated the starting material had been consumed. The reaction wasconcentrated, the residue taken up in 5 mL CH₂Cl₂ and concentrated to atan residue. The material was taken up in 1/1 Et₂O/hex and thesuspension was filtered through a fine frit. The solids were washed withadditional 1/1 Et₂O/hex and the filtrate concentrated to a tan stifffoam. The crude material was purified by flash chromatography (25 gsilica gel, 25-40% EtOAc/hex) to yield the title compound (145 mg, 83%)as a colorless glass which was a mixture of E/Z isomers: HPLC retentiontime: 4.84 min (Method A); MS (ESI+) for C₃₃H₃₉N₅O₆ m/z 602.5 (M+H)⁺; MS(ESI−) for C₃₃H₃₉N₅O₆ m/z 600.4 (M−H)¹.

Step 7. Preparation of(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-2-[(methoxyimino)methyl]-4-oxo-N-[(1R)-1-phenylethyl]azetidine-1-carboxamidetrifluoroacetate

A solution of tert-butyl(4-methoxybenzyl)(4-{[(2S,3R)-2-[(methoxyimino)methyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidin-3-yl]methyl}pyridin-2-yl)carbamate(145 mg, 0.241 mmol) in methylene chloride (4.5 mL) was cooled at 0° C.and treated with trifluoroacetic acid (1.5 mL, 20.0 mmol). The solutionwas stirred at 0° C. for 30 minutes then allowed to warm to roomtemperature. The reaction mixture was stirred for 24 h at which timeHPLC indicated the starting material had been consumed. The reactionmixture was concentrated. The residue was taken up in 10 mL CH₂Cl₂ andconcentrated to yield a tan glass. The crude material was purified byCombiFlash chromatography [30 g RediSep C-18 gold silica gel cartridge,solvent gradient: 10% acetonitrile (0.07% TFA)/water (0.1% TFA) to 100%acetonitrile (0.07% TFA)] to yield the title compound (71 mg, 59%) as awhite solid after lyophilization: (data is for the E/Z mixture) HPLCretention time: 2.88/2.93 min (Method A); MS (ESI+) for C₂₀H₂₃N₅O₃ m/z382.3 (M+H)⁺; ¹H NMR (400 MHz, MeOD) δ7.78 (1H, m) 7.48 (0.6H, d, J=6.6Hz) 7.34 (4H, m) 7.26 (1H, m) 6.96 (0.4H, d, J=4.8 Hz) 6.94 (1H, s) 6.88(1H, m) 4.94 (1H, m) 4.73 (0.4H, dd, J=4.9, 3.2 Hz) 4.43 (0.6H, dd,J=6.4, 2.9 Hz) 3.79 (1.2H, s) 3.78 (1.8H, s) 3.73 (0.6H, dt, J=8.0, 3.0Hz) 3.58 (0.4H, dt, J=7.6, 3.2 Hz) 3.21 (2H, m) 1.51 (3H, d, J=7.1 Hz)

Example 17 Scheme 9: Ethyl(2S,3R)-3-[(2-{[(hexyloxy)carbonyl]amino}pyridin-4-yl)methyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylate

Step 1. Preparation of Ethyl(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylate

A solution of ethyl(2S,3R)-3-({2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]-pyridin-4-yl}methyl)-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylate(474 mg, 0.715 mmol) in methylene chloride (9.0 mL) was cooled at 0° C.and treated dropwise with trifluoroacetic acid (3.0 mL). The solutionwas stirred at 0° C. for 60 min, followed by warming to roomtemperature. After 24 h, HPLC indicated that the starting material hadbeen consumed. The purple oil was taken up in 15 mL CH₂Cl₂ andconcentrated. The residue was dissolved in 11 mL CH₂Cl₂, treated with 10mL sat NaHCO₃ and stirred at room temperature until the bubbling ceased.The mixture was poured into a separatory funnel and the phasesseparated. The aqueous phase was washed with 20 mL CH₂Cl₂ and thecombined organic phases were dried over Na₂SO₄. The solution wasfiltered and concentrated to yield a stiff foam. The crude material waspurified by flash chromatography (35 g silica gel, 6-8% MeOH/CH₂Cl₂) toyield the title compound (254 mg, 89%) as a colorless stiff foam: HPLCretention time: 3.01 min (Method A); MS (ESI+) for C₂₁H₂₄N₄O₄ m/z 397.3(M+H)⁺; MS (ESI−) for C_(2i)H₂₄N₄O₄ m/z 395.3 (M−H)⁻.

Step 2. Preparation of Ethyl(2S,3R)-3-[(2-{[(hexyloxy)carbonyl]amino}pyridin-4-yl)methyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylate

A stirred solution of ethyl(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylate(50.0 mg, 0.126 mmol) in dry methylene chloride (0.50 mL) under nitrogenwas cooled in an ice water bath and treated with pyridine (22 μL, 0.28mmol) followed by hexyl chloroformate (23 μL, 0.14 mmol) dropwise. Thereaction mixture was stirred at 0-5° C. for 1 h followed by slow warming(over 2.5 h) to room temperature. The reaction mixture was diluted with10 mL H₂O and extracted with two 15 mL portions of CH₂Cl₂. The organicphase was washed with 15 mL portions of H₂O and brine and was dried overNa₂SO₄. The solution was concentrated to nearly colorless viscous oil.The crude material was purified by flash chromatography (25 g silicagel, 30-50% EtOAc/hex) to yield the title compound (43 mg, 66%) as acolorless glass: HPLC retention time: 4.27 min (Method A); MS (ESI+) forC₂₈H₃₆N₄O₆ m/z 525.3 (M+H)⁺; MS (ESI−) for C₂₈H₃₆N₄O₆ m/z 523.4 (M−H)⁻;¹H NMR (400 MHz, CDCl₃) δ8.21 (1H, d, J=5.1 Hz) 7.93 (1H, s) 7.73 (1H,br. s.) 7.35 (4H, m) 7.28 (1H, m) 6.89 (1H, dd, J=5.1, 1.5 Hz) 6.67 (1H,d, J=8.1 Hz) 5.03 (1H, m) 4.16 (5H, m) 3.54 (1H, ddd, J=8.3, 6.8, 2.7Hz) 3.20 (1H, m) 3.09 (1H, m) 1.70 (2H, m) 1.55 (3H, d, J=7.1 Hz) 1.37(6H, m) 1.16 (3H, t, J=7.2 Hz) 0.91 (3H, m).

Example 18 Scheme 9:(2S,3R)-3-[(2-{[(hexyloxy)carbonyl]amino}pyridin-4-yl)methyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylicacid

Step 1. Preparation of(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylate

A solution of benzyl(2S,3R)-3-({2-[(tert-butoxycarbonyl)(4-methoxybenzyl)amino]pyridin-4-yl}methyl)-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylate(344 mg, 0.507 mmol) in methylene chloride (6.4 mL) was cooled at 0° C.and treated dropwise with trifluoroacetic acid (2.1 mL, 28 mmol). Thesolution was stirred at 0° C. for 60 min, followed by warming to roomtemperature. After 24 h, HPLC indicated the reaction was complete. Thepurple oil was taken up in 15 mL CH₂Cl₂ and concentrated. The residuewas dissolved in 20 mL CH₂Cl₂, treated with 10 mL sat NaHCO₃ and stirredat room temperature until the bubbling ceased. The mixture was pouredinto a separatory funnel and the phases separated. The aqueous phase waswashed with 10 mL CH₂Cl₂ and the combined organic phases were dried overNa₂SO₄. The solution was filtered and concentrated to yield a lightyellow stiff foam. The crude material was purified by flashchromatography (30 g silica gel, (4-6% MeOH/CH₂Cl₂) to yield the titlecompound (206 mg, 89%) as a colorless stiff foam: HPLC retention time:3.48 min (Method A); MS (ESI+) for C₂₆H₂₆N₄O₄ m/z 459.3 (M+H)⁺; MS(ESI−) for C₂₆H₂₆N₄O₄ m/z 457.2 (M−H)⁻.

Step 2. Preparation of Benzyl(2S,3R)-3-[(2-{[(hexyloxy)carbonyl]amino}pyridin-4-yl)methyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylate

A stirred solution of benzyl(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylate(206 mg, 0.449 mmol) in dry methylene chloride (3.0 mL) under nitrogenwas cooled in an ice water bath and treated with pyridine (95 μL, 1.2mmol) followed by hexyl chloroformate (110 μL, 0.67 mmol) dropwise. Thereaction mixture was stirred at 0-5° C. for 1 h at which point thecooling bath was allowed to slowly warm to room temperature over 2.5 h.At this time, HPLC indicated the starting material had been consumed.The reaction mixture was diluted with 20 mL H₂O and extracted with two20 mL portions of CH₂Cl₂. The organic phase was washed with 15 mL H₂Oand brine and was dried over Na₂SO₄. The solution was concentrated to alight yellow glass. The crude material was purified by flashchromatography (30 g silica gel, 30-50% EtOAc/hex) to yield the titlecompound (242 mg, 92%) as a colorless glass: HPLC retention time: 4.51min (Method A); MS (ESI+) for C₃₃H₃₈N₄O₆ m/z 587.4 (M+H)⁺.

Step 3. Preparation of(2S,3R)-3-[(2-{[(hexyloxy)carbonyl]amino}pyridin-4-yl)methyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylicacid

A solution of benzyl(2S,3R)-3-[(2-{[(hexyloxy)carbonyl]amino}pyridin-4-yl)methyl]-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylate(242 mg, 0.412 mmol) in methanol (4.0 mL) and ethyl acetate (4.0 mL) wascarefully treated with 10% Pd-C catalyst (44 mg) The reaction flask wasevacuated and filled with hydrogen gas three times and the reaction wasstirred under an atmosphere of hydrogen for 1.5 h at which point HPLCindicated the starting material had been consumed. The reaction mixturewas filtered through a pad of solka floc and the pad was washed with 40mL 1/1 EtOAc/MeOH. The filtrate was concentrated to yield the titlecompound (197 mg, 96%) as a colorless solid: HPLC retention time: 3.77min (Method A); MS (ESI+) for C₂₆H₃₂N₄O₆ m/z 497.3 (M+H)⁺; MS (ESI−) forC₂₆H₃₂N₄O₆ m/z 495.2 (M−H)⁻; ¹H NMR (400 MHz, CDCl₃) δ9.34 (1H, br. s.)8.04 (2H, s) 7.33 (4H, s) 7.28 (1 H, m) 6.95 (1H, d, J=4.5 Hz) 6.78 (1H,d, J=7.8 Hz) 5.05 (1H, m) 4.31 (1H, br. s.) 4.15 (2H, t, J=6.7 Hz) 3.63(1H, ddd, J=8.8, 6.1, 2.8 Hz) 3.22 (1H, m) 3.09 (1H, m) 1.67 (2H, m)1.56 (3 H, d, J=6.8 Hz) 1.33 (6H, m) 0.88 (3H, m).

Example 19 Scheme 9: Ethyl(2S,3R)-3-{[2-(L-alanylamino)pyridin-4-yl]methyl}-1-{[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]carbamoyl}-4-oxoazetidine-2-carboxylatetrifluoroacetate

To a solution of benzyl(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-1-{[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]carbamoyl}-4-oxoazetidine-2-carboxylate(150 mg, 0.28 mmol) and N-(tert-butoxycarbonyl)-L-alanine (79 mg, 0.42mmol) in N,N-dimethylformamide (1.7 mL) was addedN,N-diisopropylethylamine (0.194 mL, 1.11 mmol) followed byN,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (159 mg, 0.42 mmol). After 72 h, the reaction wasdiluted with ethyl acetate and washed with sat NaHCO₃, brine, dried withanhydrous sodium sulfate, filtered and concentrated. The residue waspartially purified by flash chromatography using hexanes/ethyl acetate(30-40%) as eluent to afford benzyl(2S,3R)-3-[(2-{[N-(tert-butoxycarbonyl)-L-alanyl]amino}pyridin-4-yl)methyl]-1-{[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]carbamoyl}-4-oxoazetidine-2-carboxylate(80 mg, approximately 80% pure, 40%) as a tan solid which was usedwithout further purification. MS (ESI+) for C₃₅H₃₇F₂N₅O₉ m/z 710.2(M+H)⁺.

To a flask containing Pd/C (10%, 13 mg) was added a solution of theabove intermediate (130 mg, a combination of two lots of similar purity)in ethanol (5 mL). The mixture was stirred under 1 atmosphere of H₂ for5 h. Additional Pd/C (10%, 5 mg) was added under an atmosphere ofnitrogen and the mixture stirred an additional 3 h under 1 atmosphere ofH₂. The mixture was filtered through celite and concentrated underreduced pressure to afford(2S,3R)-3-[(2-{[N-(tert-butoxycarbonyl)-L-alanyl]amino}pyridin-4-yl)methyl]-1-{[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]carbamoyl}-4-oxoazetidine-2-carboxylicacid (100 mg) as a off white solid which was used without furtherpurification. MS (ESI+) for C₂₈H₃₁F₂N₅O₉ m/z 620.2 (M+H)⁺.

To a solution of afford(2S,3R)-3-[(2-{[N-(tert-butoxycarbonyl)-L-alanyl]amino}pyridin-4-yl)methyl]-1-{[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]carbamoyl}-4-oxoazetidine-2-carboxylicacid (100 mg, 0.16 mmol) in CH₂Cl₂ (1 mL) was added ethanol (0.28 mL,4.84 mmol) followed by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (0.120 g, 0.62 mmol) and DMAP (1 mg). After 16 h, themixture was diluted with ethyl acetate and washed with 0.1 aqueous HCl,sat NaHCO₃, brine, dried with anhydrous sodium sulfate, filtered andconcentrated. The residue was dissolved in CH₂Cl₂ (4 mL) and cooled to0-5° C. To the cooled solution was added trifluoroacetic acid (2 mL).The reaction was stirred at 0-5° C. for 30 min then at ambienttemperature for 1 h. Volatiles were removed under reduced pressure andthe residue purified by CombiFlash chromatography [30 g RediSep C-18gold silica gel cartridge, solvent gradient: 10% acetonitrile (0.07%TFA)/water (0.1% TFA) to 100% acetonitrile (0.07% TFA)] and lyophilizedto afford the title compound (24 mg, 22%) as a white solid. ¹H NMR(CD₃OD) δ1.09 (t, J=7 Hz, 3H), 1.54 (d, J=7 Hz, 3H), 1.62 (d, J=7 Hz,3H), 3.15-3.29 (m, 2H), 3.69-3.74 (m, 1H), 4.05-4.18 (m, 3H), 4.28 (d,J=3 Hz, 1H), 4.95-4.98 (m, 1H), 7.14-7.24 (overlapping m, 4H), 8.07 (brs, 1H), 8.28 (d, J=5 Hz, 1H); MS (ESI+) for C₂₅H₂₇F₂N₅O₇ m/z 548.1(M+H)⁺; HPLC retention time: 3.58 min (Method C).

Example 20 Scheme 10: Ethyl(2S,3R)-1-{[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]carbamoyl}-3-{[2-({[(isobutyryloxy)methoxy]carbonyl}amino)pyridin-4-yl]methyl}-4-oxoazetidine-2-carboxylate

Step 1. Preparation of carbonic acid chloromethyl ester 4-nitro-phenylester

To a solution of 4-nitrophenol (3.81 g, 0.027 mol) in THF (50 mL) wasadded chloromethyl chloroformate (4.00 g, 0.030 mol) followed byN,N-diisopropylethylamine (5.29 mL, 0.030 mol). The mixture was stirredfor 2 h, diluted with ethyl acetate and washed with sat NaHCO₃, brine,dried with anhydrous MgSO₄, filtered and concentrated to afford thetitle compound (6.10 g, 96%) as a yellow solid that was used withoutfurther purification. ¹H NMR (CDCl₃) δ5.88 (s, 2H), 7.44 (d, J=9 Hz,2H), 8.32 (d, J=9 Hz, 2H).

Step 2. Preparation of carbonic acid iodomethyl ester 4-nitro-phenylester

To a solution of carbonic acid chloromethyl ester 4-nitro-phenyl ester(3.00 g, 0.013 mol) in acetone (60 mL) was added sodium iodide (5.82 g,0.039 mol) and 4 Å molecular sieves (3.00 g). The mixture was heated at40° C. until judged complete by examination of an aliquot of thereaction mixture by ¹H NMR (approx. 6 h). The mixture was cooled toambient temperature and through a pad of celite. The volatiles removedat reduced pressure and the residue was dissolved in CH₂Cl₂, washed withsat NaHCO₃, water, brine, dried with anhydrous sodium sulfate, filteredand concentrated to afford the title compound (3.82 g, 91%) as a solidthat was used without further purification. ¹H NMR (CDCl₃) δ6.08 (s,2H), 7.44 (d, J=9 Hz, 2H), 8.32 (d, J=9 Hz, 2H).

Step 3. Preparation of silver 2-methylpropionate

To a solution of 2-methylpropionic acid (2.65 g, 0.030 mol) inacetonitrile (100 mL) was added silver(I) oxide (4.12 g, 0.018 mol). Theflask was protected from light and heated at 70° C. for 90 min. Themixture was cooled to ambient temperature and filtered through a pad ofcelite. The volatiles were removed in vacuo to afford the title compound(5.65 g, 96%) as a tan solid that was used without further purificationor characterization.

Step 4. Preparation of 2-methylpropionic acid4-nitro-phenoxycarbonyloxymethyl ester

To a solution of carbonic acid iodomethyl ester 4-nitro-phenyl ester(2.10 g, 6.50 mmol) in toluene (30 mL) was added silver2-methylpropanoate (2.53 g, 13.0 mmol). The mixture was heated at 55° C.until judged complete by examination of an aliquot of the reactionmixture by ¹H NMR (approx. 5 h). The mixture was cooled to ambienttemperature, filtered through a pad of celite and washed with 10%aqueous K₂CO₃ water, brine, dried with anhydrous sodium sulfate,filtered and concentrated. The residue was purified by flashchromatography using hexanes and ethyl acetate (5%) as eluent to affordthe title compound (1.51 g, 82%) as an oil: ¹H NMR (CDCl₃) δ1.25 (d, J=7Hz, 6H), 2.68 (heptet, J=7 Hz, 1H), 5.91 (s, 2H), 7.42 (d, J=9 Hz, 2 H),8.31 (d, J=9 Hz, 2H).

Step 5. Preparation of(2S,3R)-1-{[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]carbamoyl}-3-{[2-({[isobutyryloxy)methoxy]carbonyl}amino)pyridin-4-yl]methyl}-4-oxoazetidine-2-carboxylicacid

To a stirred mixture of(2S,3R)-3-[(2-aminopyridin-4-yl)methyl]-1-{[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]carbamoyl}-4-oxoazetidine-2-carboxylicacid trifluoroacetate (0.200 g, 0.36 mmol) in CH₂Cl₂ (0.5 mL) was addedchlorotrimethylsilane (0.180 mL, 1.42 mmol) andN,N-diisopropylethylamine (0.217 mL, 1.24 mmol). The mixture was heatedat 40° C. for 1 h then cooled to ambient temperature. A solution of2-methylpropionic acid 4-nitro-phenoxycarbonyloxymethyl ester (0.201 g,0.72 mmol) in CH₂Cl₂ (0.36 mL) was added followed byN,N-diisopropylethylamine (0.124 mL, 0.72 mmol). The mixture was heatedat 40° C. for 2 d then cooled to ambient temperature. The reaction wasquenched by the addition of 0.1 aqueous HCL (pH approx. 3), stirred for15 min then extracted with ethyl acetate. The combined organic layerswere washed with brine, dried with anhydrous sodium sulfate, filteredand concentrated. The residue was purified by flash chromatography usinghexanes and ethyl acetate (20%) followed by CH₂Cl₂/methanol (1-2%) aseluent to afford the title compound (0.080 g, 38%) as a glassy whitesolid: ¹H NMR (CDCl₃) δ1.19 (d, J=6 Hz, 6H), 1.56 (d, J=8 Hz, 3H), 2.61(heptet, J=7 Hz, 1H), 3.11-3.31 (m, 2H), 3.68-3.74 (m, 1 H), 4.32 (d,J=2 Hz, 1H), 5.01 (pentet, J=7 Hz, 1H), 5.83-5.88 (m, 2H), 6.75 (d, J=8Hz, 1 H), 7.04-7.08 (m, 4H), 8.06-8.12 (overlapping m, 2H), 9.79 (verybr s, 1H); MS (ESI+) for C₂₆H₂₆F₂N₄O₁₀ m/z 593.2 (M+H)⁺; HPLC retentiontime: 3.98 min (Method C).

Step 6. Preparation of ethyl(2S,3R)-1-{[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]carbamoyl}-3-{[2-({[(isobutyryloxy)methoxy]carbonyl}amino)pyridin-4-yl]methyl}-4-oxoazetidine-2-carboxylate

To a solution of(2S,3R)-1-{[(1R)-1-(2,2-difluoro-1,3-benzodioxol-5-yl)ethyl]carbamoyl}-3-{[2-({[(isobutyryloxy)methoxy]carbonyl}amino)pyridin-4-yl]methyl}-4-oxoazetidine-2-carboxylicacid (60 mg, 0.10 mmol) in CH₂Cl₂ (1 mL) was added ethanol (0.118 mL,2.02 mmol), followed by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (23 mg, 0.12 mmol) and 4-dimethylaminopyridine (0.6 mg,0.006 mmol). The mixture was stirred at ambient temperature overnight,diluted with ethyl acetate and washed with 0.25 N aqueous HCl, water,brine, dried with anhydrous sodium sulfate, filtered and concentrated.The residue was purified by flash chromatography using hexanes/ethylacetate (20-40%) as eluent to afford the title compound (35 mg, 60%) asa white solid: ¹H NMR (CDCl₃) δ1.14-1.22 (overlapping triplets, 9H),1.54 (d, J=7 Hz, 3H), 2.63 (heptet, J=7 Hz, 1H), 3.10-3.27 (m, 2 H),3.54-3.60 (m, 1H), 4.13-4.24 (overlapping m, 3H), 4.99 (pentet, J=6 Hz,1H), 5.89 (s, 2 H), 6.65 (d, J=7 Hz, 1H), 6.98-7.07 (overlapping m, 4H),8.02 (s, 1H), 8.31 (d, J=5 Hz, 1H), 9.66 (s, 1H); MS (ESI+) forC₂₈H₃₀F₂N₄O₁₀ m/z 621.1 (M+H)⁺; HPLC retention time: 5.01 min (MethodC).

Example 21 Scheme 11:(2S,3R)-3-[2-(4-methoxypheny)ethyl]-3-methyl-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylicacid

Step 1: Preparation ofbenzyl-(2S,3R)-1-[tert-butyl(dimethyl)silyl]-3-[2-(4-methoxyphenyl)ethyl]-3-methyl-4-oxoazetidine-2-carboxylate

A 50 mL 2-neck round bottom flask was charged with(2S,3R)-1-[tert-butyl(dimethyl)silyl]-3-methyl-4-oxoazetidine-2-carboxylicacid (450.0 mg, 1.849 mmol) prepared by the method of Finke, P. E., et.al., J. Med. Chem. 1995, 38, 2449. The flask was evacuated and filledwith nitrogen three times. Tetrahydrofuran (4.2 mL) was added and thesolution was cooled at 0° C. in an ice bath. A 1.45M solution of LDA inheptane/THF/ethylbenzene (2.8 mL, 1.76 mmol) was added dropwise and thesolution was stirred for 55 min at 0° C. A solution of1-bromo-2-(4-methoxyphenyl)ethane (0.54 mL, 3.5 mmol) in tetrahydrofuran(2 mL) was added dropwise and stirring was continued at 0° C. for 2 h,followed by warming the reaction mixture to rt. After 6 h at rt, thereaction mixture was diluted with 25 mL ethyl acetate and poured into 14mL of ice cold 0.5M KHSO₄ solution which was in a separatory funnel. Themixture was extracted and the phases separated. The aqueous phase waswashed with two 20 mL portions of ethyl acetate and the combined organicphase was washed with 20 mL brine. The organic phase was dried overNa₂SO₄, filtered and concentrated to yield a tan oil. The crude productwas taken up in methylene chloride (11 mL) and treated withN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (390 mg,2.03 mmol) followed by benzyl alcohol (210 μL, 2.03 mmol) and4-dimethylaminopyridine (11 mg, 0.094 mmol) and the reaction mixture wasstirred at rt for 7 h. The reaction mixture was diluted with 35 mLCH₂Cl₂ and washed with two 25 mL portions of H₂O and 20 mL brine. Theorganic phase was dried over Na₂SO₄, filtered and concentrated to yielda tan oil. The crude material was purified by flash chromatography (75 gsilica gel; 5-20% ethyl acetate/hex) to yield 168 mg of the titlecompound contaminated with an unidentified impurity as a yellow oil.Purity was 57-62% by HPLC; HPLC retention time 5.77 min (Method A).

Step 2: Preparation of benzyl(2S,3R)-3-[2-(4-methoxyphenyl)ethyl]-3-methyl-4-oxoazetidine-2-carboxylate

A solution of benzyl(2S,3R)-3-[2-(4-methoxyphenyl)ethyl]-3-methyl-4-oxoazetidine-2-carboxylate(260 mg, 0.35 mmol) in methanol (5.3 mL) was treated with acetic acid(72 μL, 1.3 mmol) followed by 0.5 M NH₄F in methanol (1.0 mL, 0.52 mmol)dropwise and the mixture was stirred at rt for 20 h at which time HPLCindicated the starting material was still present. The reaction mixturewas treated with 16 μL of HOAc and 190 μL of 0.5M NH₄F solution andallowed to stir at rt for an additional 20 h at which point HPLCindicated the reaction was complete. The reaction mixture wasconcentrated and the residue taken up in 10 mL toluene and concentrated.The process was repeated once and the residue was taken up in 25 mLCH₂Cl₂ and washed with 20 mL portions of H₂O and NaHCO₃ solution. Theorganic phase was dried over Na₂SO₄, filtered and concentrated to yielda light yellow viscous oil. The crude product was purified by flashchromatography (25 g silica gel; 20-40% ethyl acetate/hex) to yield thetitle compound (80 mg) as a colorless glass: HPLC retention time 3.81min (Method A); ¹H NMR (400 MHz, CDCl₃) δ7.39 (m, 5H), 7.11 (d, J=8.59Hz, 2H), 6.83 (m, 2H), 6.22 (br. s., 1H), 5.23 (m, 2H), 4.08 (s, 1H),3.79 (s, 3H), 2.68 (m, 2H), 2.00 (m, 2H), 1.19 (s, 3H).

Step 3: Preparation of benzyl(2S,3R)-3-[2-(4-methoxyphenyl)ethyl]-3-methyl-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylate

A solution of benzyl(2S,3R)-3-[2-(4-methoxyphenyl)ethyl]-3-methyl-4-oxoazetidine-2-carboxylate(60.0 mg, 0.170 mmol) in methylene chloride (1.8 mL) was treated withtriethylamine (95 μL, 0.679 mmol) followed by[(1R)-1-isocyanatoethyl]benzene (31 μL, 0.221 mmol) dropwise and themixture was stirred at rt for 18 h at which point HPLC indicated thestarting material had been consumed. The reaction mixture wasconcentrated, the residue taken up in 10 mL CH₂Cl₂ and concentrated to acolorless viscous oil/solid. The crude product was purified by prep TLC(20 cm×20 cm×1.0 mm prep TLC plate; 30% ethyl acetate/hex) to yield thetitle compound (72 mg) as a colorless glass: HPLC retention time 4.84min (Method A); ¹H NMR (400 MHz, CDCl₃) δ7.35 (m, 9H), 7.29 (m, 1H),7.10 (d, J=8.59 Hz, 2H), 6.84 (m, 2H), 6.71 (d, J=7.83 Hz, 1H), 5.27 (m,1H), 5.18 (m, 1H), 5.09 (m, 1H), 4.43 (s, 1H), 3.80 (s, 3H), 2.72 (m,1H), 2.62 (m, 1H), 2.04 (m, 2H), 1.57 (d, J=6.82 Hz, 3H), 1.17 (s, 3H).

Step 4: Preparation of(2S,3R)-3-[2-(4-methoxyphenyl)ethyl]-3-methyl-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylicacid

A solution of benzyl(2S,3R)-3-[2-(4-methoxyphenyl)ethyl]-3-methyl-4-oxo-1-{[(1R)-1-phenylethyl]carbamoyl}azetidine-2-carboxylate(72 mg, 0.14 mmol) in methanol (2.3 mL) and ethyl acetate (2.3 mL) wascarefully treated with 10% palladium on carbon (13 mg). The reactionflask was evacuated and filled with hydrogen gas three times and thereaction mixture was stirred at rt under an atmosphere of hydrogen for3.5 h at which point TLC (25% ethyl acetate/hex) indicated the SM hadbeen consumed. The reaction mixture was filtered through a pad of solkafloc and the pad was washed with 30 mL 1/1 ethyl acetate/MeOH. Thefiltrate was concentrated to a colorless glass that was taken up inaqueous methanol and lyophilized to yield the title compound (56 mg) asa white solid: HPLC retention time 4.19 min (Method A); MS (ESI+) forC₂₃H₂₆N₂O₅ m/z 411.2 (M+H)⁺; MS (ESI−) for C₂₃H₂₆N₂O₅ m/z 409.2 (M−H)⁻;¹H NMR (400 MHz, MeOD) δ7.35 (m, 4H), 7.26 (m, 1H), 7.15 (m, 2H), 7.11(d, J=7.83 Hz, 1 H), 6.84 (m, 2H), 4.97 (m, 1H), 4.43 (s, 1H), 3.76 (s,3H), 2.77 (m, 1H), 2.65 (m, 1H), 2.05 (m, 2H), 1.52 (d, J=7.07 Hz, 3H),1.31 (s, 3H).

Example 22 Dose Response Assay for Protease Inhibitors

-   Materials:    -   Assay buffer: 20 mM Hepes, pH 7.4; 150 mM NaCl; 0.02% Tween 20    -   Compounds: 10 mM stocks in DMSO    -   Substrate: 20 mM Glp-Pro-Arg-AMC, 25 mg/2.3 mL H₂O (store at +4°        C.) [Factor XIa, thrombin, and trypsin]        -   20 mM Pro-Phe-Arg-AMC (Bachem I-1295), 25 mg/2.2 mL H₂O            (store at +4° C.) [Factor Xa]    -   Enzyme: Factor XIa; 0.25 μM in 50% glycerol (20 μg/mL)        -   Trypsin; 0.2 μM in 50% glycerol (4.8 μg/mL)        -   Thrombin; 0.2 μM in 50% glycerol (7.34 μg/mL)        -   Factor Xa; 0.204 in 50% glycerol (9.2 μg/mL)        -   These stocks are aliquoted (˜100 μL/aliquot) and stored at            −20° C.-   Methods:-   1. Dilute the substrate to 100 μM in assay buffer (30 μL/6 mL). The    enzyme is diluted to 0.5 nM just prior to use (12 μL/6 mL for Factor    XIa; 15 μL/6 mL for all of the rest.)-   2. Pipette 50 μL of substrate into each well of the 96 well (12×8)    microtiter plate. (Column 1 is used as the 100% activity control and    receives no compound, and Column 12 is the blank and receives no    enzyme.) Add an additional 46 μL to column 2.-   3. Pipette 4 μL of each compound into the appropriate well in column    2 of the plate (unknowns assayed in triplicate, standard assayed in    duplicate). The final compound concentration will be 1/50^(th) of    the stock.-   4. Serially two-fold dilute the compound by mixing the sample in    column 2, removing 50 μL to the next well (column 3), mix and remove    to column 4, etc. until column 11. After mixing column 11, remove 50    μL and discard.-   5. Pipette 50 μL of buffer into column 12. Initiate the reaction by    adding 50 μL of enzyme solution to each well of columns 1-11 as    rapidly as possible.-   6. Read the plate in a spectrophotometer (SpectraMax) at 30° C.,    wherein each well is measured every 60 s for 30 min. For Factor Xa    assays, each well is measured every 1 min for a total of 60 min.-   7. For Factor XIa assays, the compounds are assayed, in duplicate,    at 3 different starting concentrations, 20, 2, and 0.2 μM; 1:10,    1:100, and 1:1000 dil'n of 10 mM stock. All data sets are combined    for graphing and data fitting.-   8. Data can be used both for estimation of IC₅₀ and for estimation    of K_(on).

Example 23 β-lactam Stability in Rat Plasma

-   Stock Reagents:

Normal rat plasma, stored at −80° C.

-   Protocol:-   1. Place 6 μL of each compound into 0.5 mL microcentrifuge tubes or    96-well U-bottom, polypropylene microtiter plate-   2. Place 40 μL of acetonitrile (AcN) into 0.5 mL microcentrifuge    tubes labeled 1-9-   3. Add 114 μL of plasma to each compound-   4. Sample 10 μL of compound/plasma into AcN at 2, 10, 20, 30, 60,    90, 120, 240, and 480 min of incubation at room temperature-   5. Mix each time point after adding sample and place on ice.-   6. Centrifuge time points (12,000 rpm, 3 min), remove 15 μL of the    supernatant and mix with 15 μL 0.1% TFA in a V-bottom microtiter    plate.-   7. Place microtiter plate in autosampler of HPLC and analyze on    Restek Pinnacle C18 column (2.1×100 mm).-   8. Generate extracted ion chromatogram for parent compound and    parent compound +18 (H₂O). Integrate extracted ion chromatograms    (EIC) and plot % total peak area for parent and adduct versus time.    Fit to a model of single phase exponential decay    [Y=A_(0*)exp(-k*x)+C]. T1/2 is equal to 1 n(2)/k.

Example 24 Factor XIa Enzyme Inhibition Assays

The ability of compounds of the present invention to inhibit Factor XIawas evaluated by determining the concentration of inhibitor whichresulted in a 50% reduction in enzyme activity (IC₅₀) using purifiedenzyme. Potential inhibitors of Factor XIa were evaluated using thefollowing assay.

S-2366, pyroGlu-Pro-Arg-7-methylaminocourin (AMC), available from CPCScientific, Inc., is based on the substrate pyro-Glu-Pro-Arg-pNA,available from Diapharma Group, Inc. (Columbus, Ohio), where thep-nitroanaline group is replaced with 7-methylaminocoumarin (AMC).

The final concentration of the substrate in the assay was 50 μM, and thefinal concentration of the enzyme was 0.25 nM. Inhibitors were tested byserial dilution over an appropriate range to yield a dose response curvefor determination of the inhibitors' IC₅₀ value. The assay mixture wasread every minute for 30 minutes in order to generate progress curves.The plates were read in a Spectramax m5 Multimode Plate Reader(Molecular Devices LLC, Sunnyvale, Calif.). Dose response curves werefit to Equation 1 below in which A is the maximum inhibition, B is theminimum inhibition, C is the IC₅₀, and D is the Hill coefficient.

[(A−B)/(1+(X/C)^(D))]+B   (Equation 1)

Desirable compounds have an IC₅₀ value for inhibiting Factor XIa of lessthan 1 micromolar, 100 nanomolar, 10 nanomolar, or 1 nanomolar (in orderof increasing preference).

TABLE 2 Potency, Selectivity and Stability of Exemplary CompoundsPotency Rat Plasma hFXIa FXIa Fold Selectivity Stability Compound IC₅₀(nM) FXa Thrombin Trypsin (T_(1/2); min)  1 A G G G M  2 D J G G L  3 DJ J J M  4 D J J J M  5 D J J J K  6 D J J J L  7 D J J J M  8 D J J J M 9 D J J J M  10 D J J J M  11 D J J J M  12 D J J J M  13 D J J J M  14D J J J M  15 D J J J M  16 D G G J M  17 C G G G M  18 B G G G K  19 AH G G K  20 D G G G M  21 D G G G M  22 A I H I L  23 C G G G M  24 D JG G M  25 C G G G M  26 A H I I K  27 B H G G K  28 B I I G K  29 A I IG L  31 C G G G L  32 B G G G L  33 D F G G L  34 A I I G K  35 C G G GK  36 A I I G M  37 A H I I M  38 A I I H L  39 C G G G K  40 B I I I L 42 A I I H L  43 B G G G L  44 A I I G L  45A C G G G L  45B B H H G L 46 B I G G M  47 A I I H L  48 C G G G L  49 A H I G L  50A B G I G L 50B C G G G K  51 D J J J M  52 B H I G L  53 B G G G K  54 B H I G M 55 D G G G L  56 D J J J L  57 D J J J K  68 A G I I L  88 A I I G L 89 A G I G L  91 A I I G L  92 B H I I K  95 D J J J M  96 D J J J M 97 D J J J K  98 A G I I L  99 A I I G K 100 C G G G M 102 D J J J L103 A I H G K 104 A I I I L 107 D J J J M 108 D J J J M 114 D J J J M123 C G G G L 128 D J J J L 130 C G G G L 131 D J J J M 132 C G G G K133 D J J J M 134 C G G G L 135 C G G G L 136 C J J J M 137 B H I I K138 D J J J M 139 D J J J M 140 B I I G K 144 D J J J M 145 D J J J M147 D J J J M 148 C G G G K 149 B J J J M 150 B J J J K 151 B J J J K152 D J J J M 153 D J J J M 154 D J J J M 155 C J J J M 156 C H I I L157 D J J J M 158 C J J J M 159 C J J J M 160 C J J J K 166 A H I G K168 A I I I L 169 C G H G L 170 B G G G K 171 D J J J K 175 D J J J L180 B J J J K 190 D J J J M 191 D J J J M 192 D J J J M 193 D J J J M194 D J J J M 195 D J J J M 196 D J J J M 197 C J J J M 198 C J J J M199 C J J J M 200 D J J J M For Table 2: FXIa and hFXIa refer to FactorXIa and human Factor XIa, respectively. Potency: “A” indicates <10 nM,“B” indicates 10-100 nM, “C” indicates 100-1000 nM, “D” indicates >1000nM, and “E” indicates the data is not available or has not beendetermined. Selectivity: “F” indicates <1, “G” indicates 1-500, “H”indicates 500-1000; “I” indicates >1000, and “J” indicates the data isnot available or has notbeen determined. Rat Plasma Stability: “K”indicates 0-100 min; “L” indicates >100 min; “M” indicates the data isnot available or has not been determined.

Example 25 Solubility Assays

The following procedure was used to determine the aqueous solubility ofa test compound in phosphate buffered saline (PBS —NaCl 137 mM, KCl 2.7mM, Na₂HPO₄ 8.1 mM, KH₂PO₄ 1.5 mM, pH 7.4) in 96-well plate format byHPLC-UV/VIS analysis. The test compound was prepared at 200 μM in PBSfrom a 10 mM DMSO stock solution. The final DMSO concentration was 2%.The PBS buffer samples were mixed thoroughly followed by incubation atroom temperature for 24 h. At the end of the incubation, the PBS buffersamples were centrifuged and supernatants analyzed by HPLC. The aqueoussolubility (μM) of the test compound in PBS was determined by comparingthe peak area of the principal peak in the calibration standard (200 μM)with the peak area of the corresponding peak in each of the PBS samples.The range of the assay was approximately 0.5 μM to 200 μM. The referencecompounds used in each assay were metoprolol, rifampicin, ketoconazole,phenytoin, haloperidol, simvastatin, diethylstilbestrol, and tamoxifenranking from fully soluble (200 μM) to poorly soluble (<1 μM).

Example 26 Metabolic Stability Assays

The following procedure was used to determine the stability of a testcompound in pooled liver microsomes from human (mixed gender) in 96-wellplate format. The test compound was quantified at five time points byHPLC-MS/MS analysis. The final microsomal protein concentration in theassay was 0.1 mg/mL. Each compound was tested at 0.1 μM with 0.01% DMSO,0.25% acetonitrile and 0.25% methanol. The test compound waspre-incubated with human liver microsomes in phosphate buffer (pH 7.4)for 5 min in a 37° C. shaking waterbath. The reaction was initiated byadding NADPH-generating system and incubated for 0, 15, 30, 45, and 60minutes. The reaction was stopped by transferring the incubation mixtureto acetonitrile/methanol. Samples were then mixed and centrifuged andthe supernatants used for HPLC-MS/MS analysis. Peak areas correspondingto the test compound were recorded. The compound remaining wascalculated by comparing the peak area at each time point to time zero.Four reference compounds were tested in each assay; propranolol andimipramine are relatively stable, whereas verapamil and terfenadine arereadily metabolized in human liver microsomes.

Example 27 Plasma Protein Binding Assays

The following procedure was used to determine the plasma protein bindingof a test compound in pooled plasma from human (mixed gender) viaequilibrium dialysis in a 96-well plate format. The dialysatecompartment is loaded with phosphate-buffered saline (pH 7.4) and thesample side is loaded with plasma spiked with the test compound at aconcentration of 10 μM. After loading, samples are covered and incubatedfor 4 hours at 37° C. After incubation, each compartment is sampled,diluted with acetonitrile/buffer and centrifuged. The supernatants areanalyzed by HPLC-MS/MS. The amount measured in the plasma compartmentincludes both free and bound drug, while that on the buffer siderepresents free drug only; the differences are used to calculate thepercentage plasma protein bound. Three reference compounds were testedin each assay; acebutolol, quinidine and warfarin. These compounds yieldprotein binding values that represent low, medium, and high binding tohuman plasma proteins, respectively.

TABLE 3 Plasma Protein Binding, Solubility and Metabolic Stability ofExemplary Compounds Protein Binding Aqueous Metabolic Stability Humanplasma: Solubility Human microsomes: % bound; (μM in PBS); half-life(mins) 10 μM: 4 h 10 mM: 0.1 μM: time points: incubation 4 h incubation0, 15, 30, 45, 60 min Compound @ 37 C. @ 37 C. @ 37 C. 38 P R V 88 P R V91 O R V 92 Q T Y 100 N R X 128 N R V 134 N R W 158 P U W 169 O R V Nindicates >98%; O indicates 90-98%; P indicates <90%; Q indicates thedata is not available, not detectable, or has not been determined. Rindicates >100 μM; S indicates 10-100 μM; T indicates 1-10 μM; Uindicates <1 μM V indicates >60 minutes; W indicates 30-60 minutes; Xindicates <30 minutes; Y indicates the data is not available, notdetectable, or has not been determined.

1-18. (canceled)
 19. A pharmaceutically acceptable salt of a compoundhaving the structure


20. A pharmaceutically acceptable salt of a compound having thestructure


21. A pharmaceutically acceptable salt of a compound having thestructure


22. A pharmaceutically acceptable salt of a compound having thestructure


23. A pharmaceutically acceptable salt of a compound having thestructure


24. A pharmaceutically acceptable salt of a compound having thestructure


25. The pharmaceutically acceptable salt of claim 19, wherein thepharmaceutically acceptable salt is a hydrochloride salt.
 26. Thepharmaceutically acceptable salt of claim 20, wherein thepharmaceutically acceptable salt is a hydrochloride salt.
 27. Thepharmaceutically acceptable salt of claim 21, wherein thepharmaceutically acceptable salt is a hydrochloride salt.
 28. Thepharmaceutically acceptable salt of claim 22, wherein thepharmaceutically acceptable salt is a hydrochloride salt.
 29. Thepharmaceutically acceptable salt of claim 23, wherein thepharmaceutically acceptable salt is a hydrochloride salt.
 30. Thepharmaceutically acceptable salt of claim 24, wherein thepharmaceutically acceptable salt is a hydrochloride salt.